Compositions and methods for improving carotenoid production

Abstract

The present disclosure relates to compositions and methods for producing carotenoids. The present disclosure provides genetically modified cells (e.g., microbes) that comprise a transgene encoding a ferredoxin protein, and methods of making and using said genetically modified cells. The present disclosure also provides genetically modified cells (e.g., microbes) that comprise a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP, and methods of making and using said genetically modified cells.

Description

[0001] COMPOSITIONS AND METHODS FOR

[0002] IMPROVING CAROTENOID PRODUCTION

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 571,526, filed March 29, 2024, U.S. Provisional Application Serial No. 63 / 634,685, filed April 16, 2024, and U.S. Provisional Application Serial No. 63 / 673,338, filed July 19, 2024, the content of each of which is incorporated herein by reference in its entirety.

[0005] BACKGROUND

[0006] Carotenoids are organic pigments ranging in color from yellow to red that can be naturally produced by certain organisms, including photosynthetic organisms (e.g., plants, algae, cyanobacteria), and some fungi. Carotenoids are responsible for the orange color of carrots, as well as the pink in flamingos and salmon, and the red in lobsters and shrimp.

[0007] Carotenoid pigments (e.g., P-carotene and astaxanthin) are used industrially as ingredients for food and feed stocks, both serving a nutritional function and enhancing consumer acceptability. For example, astaxanthin is widely used in salmon aquaculture to provide the orange coloration characteristic of their wild counterparts. Some carotenoids are also precursors of vitamin A. Also, carotenoids have antioxidant properties, and may have various health benefits (see, for example, Jyonouchi el al. (1991) Nutr Cancer 16(2):93-105; Giovannucci et al. (1995) J Natl Cancer Inst 87(23): 1767-1776; Miki. (1991) Pure Appl Chem 63(1): 141-146; Chew et al. (1999) Anticancer Res 19(3):1849-1853; Wang et al. (2000) Antimicrob Agents Chemother 44(9):2452-2457). Some carotenoids such as P- carotene, lycopene, and lutein are currently sold as nutritional supplements.

[0008] Although the manufacturing of carotenoids is still dominated by chemical synthesis, naturally-produced carotenoids are gaining attention and market demand. Despite the wide distribution of these pigments in plants, the low concentration of some carotenoids has hampered the development of economically feasible extraction processes. To overcome these challenges, microbial production emerges as an alternative to produce natural carotenoids. There is a need for efficient microbial systems and methods for producing natural carotenoids. SUMMARY

[0009] The present disclosure provides, among other things, genetically modified cells (e.g., microbes) comprising a transgene that encodes a ferredoxin protein. Without wishing to be bound by any particular scientific theory, the present disclosure is based at least in part on the discovery that cells (e.g., microbes, including yeasts) expressing a ferredoxin protein produced an increased level of a carotenoid (e.g., zeaxanthin). The present disclosure therefore provides, among other things, genetically modified cells (e.g., microbes) comprising a transgene encoding a ferredoxin protein, methods of making said genetically modified cells (e.g., microbes), and / or methods of producing or increasing the production of a carotenoid employing said genetically modified cells (e.g., microbes).

[0010] In some aspects, the disclosure provides a genetically modified cell comprising (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a transgene that encodes a native ferredoxin protein, wherein the transgene enhances the production of the carotenoid.

[0011] In some embodiments, the genetically modified cell is a genetically modified microbe. In some embodiments, the genetically modified microbe is a bacterium. In some embodiments, the bacterium is Escherichia coli (E. coli). In some embodiments, the genetically modified microbe is a yeast. In some embodiments, the yeast is Saccharomyces cerevisiae (S. cerevisiae), Yarrowia lipolytica, Pichia pastoris , or Kluyveromyces marxianus. In some embodiments, the yeast is not Saccharomyces cerevisiae (S. cerevisiae).

[0012] In some embodiments, the trans gene is integrated into the genome of the genetically modified cell. In some embodiments, the genetically modified cell is a yeast, and the transgene is integrated into the ARS1021 locus. In some embodiments, the transgene is not integrated into the genome of the genetically modified cell.

[0013] In some embodiments, the ferredoxin protein is a wild- type ferredoxin protein. In some embodiments, the ferredoxin protein is an engineered ferredoxin protein. In some embodiments, the ferredoxin protein is selected from APD1, ATM1, YAH1, ILV3, and NAR1. In some embodiments, the ferredoxin protein comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, and 10. In some embodiments, the transgene comprises a nucleotide sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to a nucleotide sequence selected from SEQ ID NOs: 1, 3, 5, 7, and 9. In some embodiments, the ferredoxin protein is not YAH1 or ferredoxin-3. In some embodiments, the ferredoxin protein is selected from APD1, ATM1, ILV3, and NAR1. In some embodiments, the ferredoxin protein comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 2, 4, 8, and 10. In some embodiments, the transgene comprises a nucleotide sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to a nucleotide sequence selected from SEQ ID NOs: 1, 3, 7, and 9.

[0014] In some embodiments, the genetically modified cell is a yeast, and the transgene further comprises pCCW12 promoter and / or tYOL036W terminator.

[0015] In some embodiments, the genetically modified cell comprises two or more transgenes that encode ferredoxin proteins. In some embodiments, the two or more transgenes encode the same ferredoxin protein. In some embodiments, the two or more transgenes encode different ferredoxin proteins. In some embodiments, the two or more transgenes encode different ferredoxin proteins selected from APD1, ATM1, YAH1, ILV3, and NAR1. In some embodiments, the two or more transgenes encode ferredoxin proteins comprising APD1 and ATM1, or ATM1 and YAH1.

[0016] In some embodiments, the heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid is selected from CrtE, CarRP, CarB, and / or CrtZ. In some embodiments, the CrtZ gene is not Saccharolobus solfataricus CrtZ (SsCrtZ).

[0017] In some embodiments, the cell is capable of producing an increased level of carotenoid when cultured in a culture medium comprising an iron source. In some embodiments, the iron source is iron sulfate. In some embodiments, the production of the carotenoid is enhanced by at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, or at least 1.6-fold.

[0018] In some embodiments, the carotenoid is zeaxanthin or a derivative thereof. In some embodiments, the derivative of zeaxanthin is not violaxanthin. In some embodiments, the carotenoid is selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin. In some embodiments, the carotenoid is zeaxanthin or astaxanthin.

[0019] In some aspects, the disclosure provides a genetically modified cell comprising a transgene that encodes a native ferredoxin protein, wherein the genetically modified cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the native ferredoxin protein, optionally wherein the genetically modified cell is a genetically modified microbe. In some aspects, the disclosure provides a genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the ferredoxin protein is not YAH1 or ferredoxin-3, optionally wherein the genetically modified cell is a genetically modified microbe. In some embodiments, the ferredoxin protein is selected from APD1, ATM1, ILV3, and NARl.

[0020] In some aspects, the disclosure provides a genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the ferredoxin protein, wherein the carotenoid is not violaxanthin, optionally wherein the genetically modified cell is a genetically modified microbe. In some embodiments, the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin. In some embodiments, the carotenoid is zeaxanthin or astaxanthin.

[0021] In some aspects, the disclosure provides a method of producing a carotenoid, comprising culturing a genetically modified cell disclosed herein in a culture medium. In some embodiments, the method comprises culturing a genetically modified cell disclosed herein in a culture medium in a batch fermentation. In some embodiments, the culture medium comprises an iron source. In some embodiments, the iron source is iron sulfate. In some embodiments, the method further comprises extracting the carotenoid from the genetically modified cell. In some embodiments, the carotenoid is zeaxanthin or a derivative thereof. In some embodiments, the derivative of zeaxanthin is not violaxanthin. In some embodiments, the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin. In some embodiments, the carotenoid is zeaxanthin or astaxanthin.

[0022] In some aspects, the disclosure provides a method of increasing production of zeaxanthin or a derivative thereof, wherein the derivative of zeaxanthin is not violaxanthin, comprising culturing a genetically modified cell disclosed herein in a culture medium.

[0023] In some aspects, the disclosure provides a method of making a genetically modified cell disclosed herein, comprising contacting the cell with a nucleic acid encoding a ferredoxin protein. In some embodiments, the nucleic acid is an expression vector.

[0024] In some aspects, the disclosure provides a method of increasing a carotenoid production in a cell, comprising contacting the cell with an agent that increases gene copy number, expression, and / or activity of ferredoxin, wherein the ferredoxin is not YAH1 or ferredoxin-3, optionally wherein the genetically modified cell is a genetically modified microbe. In some embodiments, the ferredoxin is selected from APD1, ATM1, ILV3, and NAR1.

[0025] In some aspects, the disclosure provides a method of increasing production of a carotenoid in a cell, wherein the carotenoid is not violaxanthin, comprising contacting the cell with an agent that increases gene copy number, expression, and / or activity of ferredoxin, optionally wherein the genetically modified cell is a genetically modified microbe. In some embodiments, the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin. In some embodiments, the carotenoid is zeaxanthin or astaxanthin. In some embodiments, the agent is a nucleic acid, a protein, and / or a small molecule. In some embodiments, the agent is a nucleic acid encoding a ferredoxin protein.

[0026] The present disclosure also provides, among other things, genetically modified cells (e.g., microbes) comprising a transgene that encodes a carotenoid binding protein (CBP) or a genetic modification capable of increasing the expression of a CBP. Without wishing to be bound by any particular scientific theory, the present disclosure is based at least in part on the discovery that cells (e.g., microbes, including yeasts) expressing a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP produced an increased level of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof). The present disclosure therefore provides, among other things, genetically modified cells (e.g., microbes) comprising a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP, methods of making said genetically modified cells (e.g., microbes), and / or methods of producing or increasing the production of a carotenoid employing said genetically modified cells (e.g., microbes).

[0027] In some aspects, the disclosure provides a genetically modified cell comprising (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a transgene that encodes a heterologous carotenoid binding protein (CBP), wherein the transgene enhances the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof.

[0028] In some aspects, the disclosure provides a genetically modified cell comprising (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a genetic modification capable of increasing the expression of a carotenoid binding protein (CBP), wherein the genetic modification enhances the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof.

[0029] In some embodiments, the genetically modified cell is a genetically modified microbe. In some embodiments, the genetically modified microbe is a bacterium. In some embodiments, the bacterium is Escherichia coli (E. coli). In some embodiments, the genetically modified microbe is a yeast. In some embodiments, the yeast is Saccharomyces cerevisiae (S. cerevisiae), Yarrowia lipolytica, Pichia pastoris, or Kluyveromyces marxianus. In some embodiments, the yeast is not Saccharomyces cerevisiae (S. cerevisiae). In some embodiments, the transgene is integrated into the genome of the genetically modified cell. In some embodiments, the genetically modified cell is a yeast, and the transgene is integrated into the ARS511 locus. In some embodiments, the transgene is not integrated into the genome of the genetically modified cell.

[0030] In some embodiments, the CBP is a wild-type CBP. In some embodiments, the CBP is an engineered CBP. In some embodiments, the CBP is selected from the steroidogenic acute regulatory lipid transfer (START) protein family or the Aster family of proteins. In some embodiments, the CBP is selected from the steroidogenic acute regulatory lipid transfer (START) protein family. In some embodiments, the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog. In some embodiments, the CBP is Bombyx mori CBP. In some embodiments, the CBP comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 12. In some embodiments, the transgene comprises a nucleotide sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 11. In some embodiments, the CBP is selected from the Aster family of proteins. In some embodiments, the CBP is selected from Aster-A, Aster -B, and Aster -C, or homologs thereof. In some embodiments, the CBP is human Aster- A, or a human Aster-A homolog. In some embodiments, the CBP comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 14. In some embodiments, the transgene comprises a nucleotide sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 13. In some embodiments, the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), Homarus gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof.

[0031] In some embodiments, the genetically modified cell is a yeast, and the transgene further comprises pGAL7 promoter and / or tHSP26 terminator. In some embodiments, the heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid is selected from CrtE, CarRP, CarB, and / or CrtZ. In some embodiments, the production of zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is enhanced by at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6- fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold. In some embodiments, the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and betacryptoxanthin. In some embodiments, the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is zeaxanthin or astaxanthin.

[0032] In some aspects, the disclosure provides a genetically modified cell comprising a transgene that encodes a heterologous carotenoid binding protein (CBP), wherein the genetically modified cell is capable of producing an increased level of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof, compared to a cell having the same genetic background but without the transgene encoding the heterologous carotenoid binding protein (CBP), optionally wherein the genetically modified cell is a genetically modified microbe. In some aspects, the disclosure provides a genetically modified cell comprising a transgene that encodes a heterologous carotenoid binding protein (CBP), wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof, optionally wherein the genetically modified cell is a genetically modified microbe. In some embodiments, the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog, or human Aster-A or a human Aster-A homolog.

[0033] In some aspects, the disclosure provides a method of producing (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof, comprising culturing a genetically modified cell disclosed herein in a culture medium. In some embodiments, the method comprises culturing a genetically modified cell disclosed herein in a culture medium in a batch fermentation. In some embodiments, the culture medium comprises an iron source. In some embodiments, the iron source is iron sulfate. In some embodiments, the method further comprises extracting the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof from the genetically modified cell. In some embodiments, the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin. In some embodiments, the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is zeaxanthin or astaxanthin.

[0034] In some aspects, the disclosure provides a method of making a genetically modified cell disclosed herein, comprising contacting the cell with a nucleic acid encoding a heterologous carotenoid binding protein (CBP). In some embodiments, the nucleic acid is an expression vector.

[0035] In some aspects, the disclosure provides a method of increasing the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof in a cell, comprising contacting the cell with an agent that increases gene copy number, expression, and / or activity of a heterologous carotenoid binding protein (CBP), wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof, optionally wherein the genetically modified cell is a genetically modified microbe. In some embodiments, the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog, or human Aster- A or a human Aster- A homolog. In some embodiments, the agent is a nucleic acid, a protein, and / or a small molecule. In some embodiments, the agent is a nucleic acid encoding a heterologous CBP.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGs. 1A-1F provide plots depicting production of zeaxanthin (FIGs. 1A, 1C, and IE) and beta-carotene (FIGs. IB, ID, and IF) by parent S. cerevisiae strains (STR2820, STR3142, and STR3145) or child strains with overexpression of individual ferredoxin genes in an initial 96-well plate screening.

[0038] FIGs. 2A-2B provide plots depicting production of zeaxanthin (FIG. 2A) and betacarotene (FIG. 2B) by a parent 5. cerevisiae strain (STR3334) or child strains with overexpression of individual or combinations of ferredoxin genes in an initial 96-well plate screening.

[0039] FIG. 3A-3B provide plots depicting production of zeaxanthin (FIG. 3A) and betacarotene (FIG. 3B) by parent S. cerevisiae strains (STR3142, STR3145, and STR3334) or child strains with overexpression of ferredoxin genes in batch fermentation.

[0040] FIG. 4 provides plots depicting increased zeaxanthin production by parent S. cerevisiae strains (STR3072, STR3204, and STR3334) or child strains with overexpression of ferredoxin genes (STR3383, STR3386) with supplemented iron in media in batch fermentation.

[0041] FIGs. 5A-5L provide plots depicting production of zeaxanthin (FIGs. 5A, 5C, 5E, 5G, 51, and 5K) and beta-carotene (FIGs. 5B, 5D, 5F, 5H, 5J, and 5L) by parent S. cerevisiae strains (STR3072, STR3496, STR3519, STR3780, STR4002, and STR4114) or child strains with overexpression of individual ferredoxin genes in an initial 96-well plate screening.

[0042] FIG. 6 provides a plot depicting production of beta-carotene by parent 5. cerevisiae strain STR4344 or child strain STR4460 with overexpression of YAH1 in 96-well plate screening.

[0043] FIGs. 7A-7D provide plots depicting production of astaxanthin (FIGs. 7A and 7C) and beta-carotene (FIGs. 7B and 7D) by parent S. cerevisiae strains (STR4873 and STR5249) or child strains with overexpression of YAH1 in 96-well plate screening.

[0044] FIGs. 8A-8D provide plots depicting production of zeaxanthin (FIGs. 8 and 8C) and beta-carotene (FIGs. 8B and 8D) by parent S. cerevisiae strains (STR4002 and STR4114) or child strains with overexpression of APD1 in batch fermentation.

[0045] FIG. 9 provides plots depicting cell density and zeaxanthin production by a parent .S'. cerevisiae strain (STR3072), a child strain with overexpression of BmCBP (STR3519), or a control S. cerevisiae strain CEN.PK 113-7D in an initial 96-well plate screening.

[0046] FIG. 10 provides a plot depicting zeaxanthin production by parent S. cerevisiae strains (STR3072 and STR3575) or child strains with overexpression of BmCBP (STR3519 and STR3668) in batch fermentation. STR3519 is the child strain of STR3072. STR3668 is the child strain of STR3575.

[0047] FIG. 11 provides a plot depicting zeaxanthin production by a S. cerevisiae child strain with overexpression of BmCBP (STR3992) in batch fermentation, and zeaxanthin production by strains derived from STR3992 where BmCBP was replaced with human Aster-A (STR4138), truncated human ApoB (STR4140), or H. gammarus crustacyanin A2 subunit (STR4232).

[0048] FIG. 12 provides a plot depicting zeaxanthin production by a S. cerevisiae child strain with overexpression of BmCBP (STR3992) in batch fermentation, and zeaxanthin production by a strain derived from STR3992 where BmCBP was swapped with human ApoB (STR4336).

[0049] DETAILED DESCRIPTION

[0050] The present disclosure includes, among other things, genetically modified cells (e.g., microbes) comprising a transgene that encodes a ferredoxin protein. The present disclosure further includes, among other things, methods of producing or producing at an increased level of a carotenoid employing said genetically modified cells (e.g., microbes), and methods of making said genetically modified cells (e.g., microbes).

[0051] As noted above, most carotenoids used in industry are produced by chemical synthesis. Although microbial biosynthesis of carotenoids was demonstrated in microbes engineered to express biosynthetic enzymes involved in the carotenoid pathway, applications of cofactors in the carotenoid reaction remain largely unexplored. The synthesis of carotenoids and xanthophylls through the carotenoid biosynthetic pathway required cofactors 2Fe-2S ferredoxin proteins as electron donors for the reaction. However, ensuring that cofactors are properly balanced is a common issue for producing a metabolite product in a recombinant host. The present disclosure provides genetically modified cells (e.g., microbes) and methods of employing said cells (e.g., microbes) to bioproduce a carotenoid, thus addressing the limitations of pre-existing chemical and extraction-based methodologies. The genetically modified cells (e.g., microbes) and their uses disclosed herein are particularly advantageous because they provide an avenue to balance the cofactors 2Fe-2S ferredoxin proteins to optimize carotenoid production and drive more flux towards desired products.

[0052] The present disclosure also includes, among other things, genetically modified cells (e.g., microbes) comprising a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP. The present disclosure further includes, among other things, methods of producing or producing at an increased level of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof) employing said genetically modified cells (e.g., microbes), and methods of making said genetically modified cells (e.g., microbes).

[0053] As noted above, most carotenoids used in industry are produced by chemical synthesis. Although microbial biosynthesis of carotenoids was demonstrated in microbes engineered to express biosynthetic enzymes involved in the carotenoid pathway, there are limitations to using microbes for producing rigid, lipophilic carotenoids such as zeaxanthin, which may not diffuse through the lipid bilayer of the plasma membrane but instead become embedded in membrane bound organelles, leading to toxicity associated with the accumulation of carotenoids. The present disclosure provides genetically modified cells (e.g., microbes) and methods of employing said cells (e.g., microbes) to bioproduce and store a carotenoid, thus addressing the limitations of pre-existing biological or chemical and extraction-based methodologies. Without wishing to be bound by any particular scientific theory, the genetically modified cells (e.g., microbes) and their uses disclosed herein are particularly advantageous because they provide an avenue to increase the cellular storage capacity of carotenoids to optimize carotenoid production and drive more flux towards desired products. Definitions

[0054] As used herein, “a”, “an”, and “the” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” discloses embodiments of exactly one element and embodiments including more than one element.

[0055] As used herein, term “about”, when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0056] As used herein, the term “bioproduction” is intended to mean production of a compound (e.g., carotenoid) by way of biological or enzymatic synthesis (as opposed to chemical synthesis). In some embodiments, bioproduction may be performed by a transgenic organism or microbe that has been engineered to express enzymes involved in the biological synthesis of a compound of interest (e.g., carotenoid).

[0057] As used herein, the term “carotenoid” is understood in the art to refer to a structurally diverse class of pigments derived from isoprenoid pathway intermediates. The commitment step in carotenoid biosynthesis is the formation of phytoene from geranylgeranyl pyrophosphate. Carotenoids can be acyclic or cyclic, and may or may not contain oxygen, so that the term carotenoids include both carotenes and xanthophylls. In general, carotenoids are hydrocarbon compounds having a conjugated polyene carbon skeleton formally derived from the five-carbon compound IPP, including triterpenes (C30 diapocarotenoids) and tetraterpenes (C40 carotenoids) as well as their oxygenated derivatives and other compounds that are, for example, C35, C50, Ceo, C70, Cso in length or other lengths. Many carotenoids have strong light absorbing properties and may range in length in excess of C200. C30 diapocarotenoids typically consist of six isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the center of the molecule so that the two central methyl groups are in a 1,6-positional relationship and the remaining non-terminal methyl groups are in a 1,5- positional relationship. Such C30 carotenoids may be formally derived from the acyclic C30H42 structure, having a long central chain of conjugated double bonds, by: (i) hydrogenation (ii) dehydrogenation, (iii) cyclization, (iv) oxidation, (v) esterification / glycosylation, or any combination of these processes. C40 carotenoids typically consist of eight isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the center of the molecule so that the two central methyl groups are in a 1,6-positional relationship and the remaining non-terminal methyl groups are in a 1,5- positional relationship. Such C40 carotenoids may be formally derived from the acyclic C40H56 structure, having a long central chain of conjugated double bonds, by (i) hydrogenation, (ii) dehydrogenation, (iii) cyclization, (iv) oxidation, (v) esterification / glycosylation, or any combination of these processes. The class of C40 carotenoids also includes certain compounds that arise from rearrangements of the carbon skeleton, or by the removal of part of this structure. More than 600 different carotenoids have been identified in nature. Carotenoids include but are not limited to: antheraxanthin, adonirubin, adonixanthin, astaxanthin, canthaxanthin, capsorubin, -cryptoxanthin, a- carotene, -carotene, 5-carotene, e-carotene, echinenone, 3 -hydroxy echinenone, 3'- hydroxyechinenone, y-carotene, lycopene, 4-keto-y-carotene, ^-carotene, a-cryptoxanthin, deoxyflexixanthin, diatoxanthin, 7,8-didehydroastaxanthin, didehydrolycopene, fucoxanthin, fucoxanthinol, isorenieratene, P-isorenieratene, lactucaxanthin, lutein, lycopene, myxobactone, neoxanthin, neurosporene, hydroxyneurosporene, peridinin, phytoene, rhodopin, rhodopin glucoside, 4-keto-rubixanthin, siphonaxanthin, spheroidene, spheroidenone, spirilloxanthin, torulene, 4-keto-torulene, 3-hydroxy-4-keto-torulene, uriolide, uriolide acetate, violaxanthin, zeaxanthin-P-diglucoside, zeaxanthin, rhodoxanthin, and C30 carotenoids. Additionally, carotenoid compounds include derivatives of these molecules, which may include hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups. Further, included carotenoid compounds include esters (e.g., fatty acid ester), ethers (e.g., glycosides) and sulfate derivatives (e.g., esterified xanthophylls). In some embodiments, the carotenoid is zeaxanthin or a derivative thereof, e.g., zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin.

[0058] As used herein, the term “biosynthetic enzymes’’ refers to enzymes involved in the production of a particular compound or a class of compounds. In some embodiments, biosynthetic enzymes catalyze particular steps in a synthesis pathway that produces a particular compound or a class of compounds. In some embodiments, the term “biosynthetic enzymes” may also encompass enzymes that do not themselves catalyze synthetic reactions in a synthesis pathway, but that regulate the expression and / or activity of other enzymes that do so. In some embodiments, the biosynthetic enzymes are carotenoid biosynthetic enzymes that are involved in the production of one or more carotenoids. Carotenoid biosynthetic enzymes include but are not limited to, for example, geranylgeranyl diphosphate synthase, bifunctional lycopene cyclase / phytoene synthase, phytoene synthase, phytoene dehydrogenase (or desaturase), lycopene cyclase, carotenoid ketolase (e.g., P-carotene ketolase), carotenoid hydroxylase (e.g., P-carotene hydroxylase), astaxanthin synthase, carotenoid epsilon hydroxylase, lycopene cyclase (beta and epsilon subunits), carotenoid glucosyltransferase, and acyl CoA:diacyglycerol acyltransferase, and alcohol acetyl transferase. In some embodiments, the carotenoid biosynthetic enzymes include geranylgeranyl diphosphate synthase (e.g., CrtE), bifunctional lycopene cyclase / phytoene synthase (e.g., CrtYB, CarRA, or CarRP), phytoene desaturase (e.g., Crtl or CarB), P-carotene hydroxylase (e.g., CrtZ, CHYb), and / or P-carotene ketolase (e.g., CrtW).

[0059] As used herein, the term “carotenoid binding protein” or “CBP” refers to a protein that is capable of binding a carotenoid. In some embodiments, a carotenoid binding protein may bind and transport carotenoids, cholesterol, bile acids, steroid hormones, and / or ceramides. Carotenoid binding proteins include but are not limited to, for example, steroidogenic acute regulatory lipid transfer (START) protein family proteins, Aster family proteins, lipoproteins (e.g., very low density lipoproteins, low density lipoproteins, and high density lipoproteins), the orange carotenoid protein (OCP) protein family proteins, helical carotenoid proteins (HCPs), and chlorophyll-binding proteins (e.g., chlorophyll a / b-binding proteins). In some embodiments, a carotenoid binding protein may comprise a START domain or a START-like domain.

[0060] The term “genetically modified cell” refers to a genetically modified cell (e.g., a prokaryotic cell or an eukaryotic cell) wherein the modification can be selected from e.g., increased expression of a gene, inhibited expression of a gene, knockout of a gene, introduction of new gene(s), introduction of mutant gene(s), or mutation / genetic alteration of gene(s), wherein the increased expression or inhibited expression of a gene can be achieved by using common techniques in the art, such as gene deletion, changed gene copy number, changed gene promoter (e.g., by using a strong or weak promoter), etc. A genetically modified cell may also include a cell that has been isolated. In some embodiments, a genetically modified cell is a transgenic cell capable of producing high levels of a compound or biomolecule of interest. In some embodiments, the genetically modified cell refers to a genetically modified microbe. An example of a microbe herein may be a microbial cell (e.g., bacteria, yeast, fungi, etc.).

[0061] As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool). For the purposes of this disclosure, a gene (e.g., a gene encoding a ferredoxin protein or a gene encoding a CBP) or a protein (e.g., a ferredoxin protein or a CBP) may comprise a nucleic acid sequence or an amino acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to about 100% sequence identity or homology with a reference or “parent” sequence.

[0062] As used herein with respect to a molecule such as a nucleic acid or polypeptide, or form thereof, “level” is used to refer to a measure indicative of an amount, concentration, ratio, or activity of the molecule, e.g., in a particular context such as a tissue, sample, organism, or a context representative thereof. An amount can be, for example, a mass or number of molecules. A concentration can be an amount relative to a context value, e.g., per a unit of mass or volume. A ratio can be a relationship between two values, such as an experimental value and a reference control value. Activity can be a measure of a function associated with a molecule, and can in various instances be measured relative to a context value, e.g., per a unit of mass or volume. Those of skill in the art will appreciate that the metric by which a level is expressed can vary depending, e.g., on the assay and purpose. Those of skill in the art will further appreciate that metrics such as amount, concentration, ratio, and activity are often interrelated and / or qualitatively or quantitatively informative of each other.

[0063] As used herein, in its broadest sense, the term “nucleic acid” refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, the term nucleic acid refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside), and in some embodiments refers to a polynucleotide chain including a plurality of individual nucleic acid residues. A nucleic acid can be or include DNA, RNA, or any combination thereof. A nucleic acid can include natural nucleic acid residues, nucleic acid analogs, and / or synthetic residues. In some embodiments, a nucleic acid includes natural nucleotides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a nucleic acid is or includes of one or more nucleotide analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5- methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2- aminoadenosine, C5- bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, a nucleic acid includes one or more genes. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid can include one or more peptide nucleic acids, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid includes one or more modified sugars (e.g., 2'- fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid is or includes at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, a nucleic acid is partly or wholly single stranded, or partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence including at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0064] As used herein, the term “transgene” refers to a nucleic acid (e.g., DNA or RNA) sequence encoding a protein or RNA (e.g., a functional non-coding RNA). In some embodiments, the transgene comprises an open reading frame encoding a protein or RNA (e.g., a functional non-coding RNA). A transgene may be isolated from an organism and introduced into a different organism of the same or different species to produce the transgene product (e.g., the protein or RNA). Some non-limiting examples of transgenes according to the present disclosure are transgenes encoding ferredoxin proteins or CBPs. As used herein, in the context of a DNA encoding a protein, the term transgene may or may not include untranscribed flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, terminators, promoters, or enhancers.

[0065] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to a compound comprised of amino acid residues covalently linked by peptide bonds, or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. In some embodiments, a peptide, a polypeptide, or a protein (e.g., a wild-type ferredoxin protein or a wild-type CBP) has an amino acid sequence that occurs in nature. In some embodiments, a peptide, a polypeptide, or a protein (e.g., an engineered ferredoxin protein or an engineered CBP) has an amino acid sequence that does not occur in nature. In some embodiments, a peptide, a polypeptide, or a protein (e.g., a ferredoxin protein or a CBP) may include natural amino acids, non-natural amino acids, or both. In some embodiments, a peptide, a polypeptide, or a protein (e.g., a native ferredoxin protein or a native CBP) is normally found in or made by an organism (e.g., a microbe). In some embodiments, a peptide, a polypeptide, or a protein (e.g., a heterologous ferredoxin protein or a heterologous CBP) is not normally found in or made by an organism (e.g., a microbe).

[0066] Genetically Modified Cells In some aspects, provided herein is a genetically modified cell (e.g., microbe) comprising a trans gene that encodes a ferredoxin protein. In some embodiments, the genetically modified cell (e.g., microbe) provided herein expresses the ferredoxin protein.

[0067] Ferredoxins are iron-sulfur proteins that mediate electron transfer in a range of metabolic reactions. For example, they can serve as electron donors in reactions involved in the bioproduction of carotenoids.

[0068] The genetically modified cell (e.g., microbe) described herein may or may not express an endogenous ferredoxin protein. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene that encodes a wild-type ferredoxin protein. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene that encodes an engineered ferredoxin protein. The engineered ferredoxin protein may be a functionally active fragment of a wild-type ferredoxin protein, a ferredoxin protein fused with a tag (e.g., a peptide tag, such as a Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, or V5 tag) or a reporter protein (e.g., a GFP protein, a luciferase protein, etc.), and / or a mutated ferredoxin protein with one or more amino acid deletions, insertions, and / or substitutions. In preferred embodiments, the mutated ferredoxin protein maintains or increases its activity in mediating electron transfer.

[0069] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that is native to the cell (e.g., microbe) , i.e., from the same species as the cell (e.g., microbe) . In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that is heterologous to the cell (e.g., microbe) , i.e., from a different species as the cell (e.g., microbe) .

[0070] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a bacterial ferredoxin protein. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a plant ferredoxin protein, e.g., FD3 or RFNR1 from A. thaliana. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a yeast ferredoxin protein. In some embodiment, the yeast ferredoxin protein is selected from APD1 (actin patches distal 1), ATM1 (mitochondrial inner membrane ATP -binding cassette transporter 1), YAH1 (yeast adrenodoxin homolog 1), ILV3 (mitochondrial dihydroxyacid dehydratase), NAR1 (nuclear architecture-related protein 1), AIM32 (altered inheritance of mitochondria protein 32), ARH1 (adrenodoxin reductase homolog 1), DRE2 (Fe-S cluster assembly protein DRE2), and TAH18 (NADPH-dependent diflavin oxidoreductase 1). In some embodiments, the yeast ferredoxin protein is selected from APD1, ATM1, ILV3, NAR1, AIM32, ARH1, DRE2, and TAH18. In some embodiments, the yeast ferredoxin protein is selected from APD1, ATM1, ILV3, and NAR1.

[0071] In some embodiments, the genetically modified cell comprises a transgene encoding a ferredoxin protein that is not YAH1 or plant ferredoxin-3. In some embodiments, the genetically modified cell may comprise two or more (e.g., 2, 3, 4, or 5) transgenes that encode ferredoxin proteins as disclosed herein. In some embodiments, the two or more (e.g., 2, 3, 4, or 5) transgenes encode the same ferredoxin protein, e.g., a single ferredoxin protein selected from APD1 , ATM1 , YAH1 , ILV3, NAR1 , AIM32, ARH1 , DRE2, and TAH18. In some embodiments, the two or more (e.g., 2, 3, 4, or 5) transgenes encode different ferredoxin proteins, e.g., different ferredoxin proteins selected from APD1, ATM1, YAH1, ILV3, NAR1, AIM32, ARH1, DRE2, and TAH18. In some embodiments, each of the two or more (e.g., 2, 3, 4, or 5) transgenes encode a different ferredoxin protein. In some embodiments, the two or more (e.g., 2, 3, 4, or 5) transgenes encode different ferredoxin proteins selected from APD1, ATM1, YAH1, ILV3, and NAR1. In some embodiments, the two or more (e.g., 2, 3, 4, or 5) transgenes encode different ferredoxin proteins selected from APD1, ATM1, and YAH1. In some embodiments, the two or more (e.g., 2, 3, 4, or 5) transgenes encode APD1 and ATM1, or ATM1 and YAH1.

[0072] Exemplary nucleic acid and protein sequences of the ferredoxin proteins encompassed within the scope of compositions-of-matter and methods of the present disclosure are shown in Table 1 below.

[0073] Table 1. Exemplary DNA and Amino Acid Sequences of Ferredoxins

[0074] As is well-known to those skilled in the art, polypeptides having substantial sequence similarities can have the same or similar catalytic and / or functional activity. Accordingly, in some embodiments, a derivative, equivalent, variant, fragment, or mutant of a ferredoxin protein described herein or fragment thereof may also suitable for the methods and compositions provided herein.

[0075] There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code. An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed. Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0076] For a ferredoxin amino acid sequence, corresponding nucleotide sequences that can encode the ferredoxin protein can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Therefore, description and / or disclosure of a ferredoxin amino acid sequence herein should be considered to also include description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0077] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that may comprise, or consist of, the amino acid sequence of SEQ ID NO: 2. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0078] In some embodiments, the ferredoxin protein has an amino acid sequence that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 300, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 or 316 consecutive amino acids of SEQ ID NO: 2.

[0079] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that comprises an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 2.

[0080] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that may comprise, or consist of, the amino acid sequence of SEQ ID NO: 4. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 4.

[0081] In some embodiments, the ferredoxin protein has an amino acid sequence that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,

[0082] 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530,

[0083] 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 655, 660, 665, 670, 675, 680, 685 or 690 consecutive amino acids of SEQ ID NO: 4.

[0084] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that comprises an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 4.

[0085] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that may comprise, or consist of, the amino acid sequence of SEQ ID NO: 6. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 6.

[0086] In some embodiments, the ferredoxin protein has an amino acid sequence that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 125, 130, 135, 140, 145, 150, 155, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or 172 consecutive amino acids of SEQ ID NO: 6.

[0087] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that comprises an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 6.

[0088] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that may comprise, or consist of, the amino acid sequence of SEQ ID NO: 8. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0089] In some embodiments, the ferredoxin protein has an amino acid sequence that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, or 585 consecutive amino acids of SEQ ID NO: 8.

[0090] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that comprises an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 8.

[0091] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that may comprise, or consist of, the amino acid sequence of SEQ ID NO: 10. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0092] In some embodiments, the ferredoxin protein has an amino acid sequence that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,

[0093] 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 481, 482, 483, 484, 485,

[0094] 486, 487, 488, 489, 490, or 491 consecutive amino acids of SEQ ID NO: 10.

[0095] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a ferredoxin protein that comprises an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 10.

[0096] In some embodiments, provided herein is a genetically modified cell (e.g., microbe) comprising a transgene that encodes a ferredoxin protein, and the transgene encoding the ferredoxin protein may comprise, or consist of, the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the trans gene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 1.

[0097] In some embodiments, provided herein is a genetically modified cell (e.g., microbe) comprising a transgene that encodes a ferredoxin protein, and the transgene encoding the ferredoxin protein may comprise, or consist of, the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the trans gene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 3.

[0098] In some embodiments, provided herein is a genetically modified cell (e.g., microbe) comprising a transgene that encodes a ferredoxin protein, and the transgene encoding the ferredoxin protein may comprise, or consist of, the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the trans gene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 5.

[0099] In some embodiments, provided herein is a genetically modified cell (e.g., microbe) comprising a transgene that encodes a ferredoxin protein, and the transgene encoding the ferredoxin protein may comprise, or consist of, the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the trans gene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 7.

[0100] In some embodiments, provided herein is a genetically modified cell (e.g., microbe) comprising a transgene that encodes a ferredoxin protein, and the transgene encoding the ferredoxin protein may comprise, or consist of, the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the trans gene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 9.

[0101] For the purposes of the present disclosure, any of the foregoing ferredoxin proteins can be expressed in a host cell, transgenic cell or modified cell (e.g., a host microbe, transgenic microbe, or modified microbe), and any of the foregoing nucleic acids may incorporated into a host cell, transgenic cell or modified cell (e.g., a host microbe, transgenic microbe, or modified microbe)in order to produce carotenoids according to the disclosed methods.

[0102] The genetically modified cell that comprises a transgene encoding a ferredoxin protein may be a prokaryote or a eukaryote. For example, the genetically modified cell may be a microbe. The genetically modified cell (e.g., microbe) may be a bacterium, an archaeon, a protozoa, an alga, or a fungus.

[0103] In some embodiments, the genetically modified cell (e.g., microbe) is a prokaryote. Model prokaryotic systems that may be utilized as a genetically modified cell (e.g., microbe) include but are not limited to Escherichia coli (E. coli), an Acinetobacter species, a Pseudomonas species, a Streptomyces species, and a Mycobacterium species. Additional suitable prokaryotic expression systems include, but are not limited to, Klebsiella, Lactococcus, Mannheimia, Corynebacterium, Vibrio, and Bacillis. In some embodiments, the genetically modified cell (e.g., microbe) is a bacterium, e.g., Escherichia coli (E. coli).

[0104] In some embodiments, the genetically modified cell (e.g., microbe) is a eukaryote. Model eukaryotic systems that may be utilized as a genetically modified cell (e.g., microbe) include, but are not limited to, Saccharomyces cerevisiae (S. cerevisiae) or other yeast species (e.g., Yarrowia lipolytica, Pichia pastoris, or Kluyveromyces marxianus)-, a filamentous fungus, optionally selected from an Aspergillus species and a Trichoderma species; an alga, optionally selected from Dunaliella salina, Haemaiococcus lacustris, Botryococcus braunii, Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp., and Tetraselmis suecia', and an amoeba, which is optionally Dictyostelium discoideum. Additional suitable eukaryotic expression systems include, but are not limited to, Pichia pastoris, Rhodosporidium toruloides, Aspergillus (oryzae, nidulans, or niger), Trichoderma reesei, and Penicillium chrysogenum, etc. In some embodiments, the genetically modified cell (e.g., microbe) is a yeast, e.g., Saccharomyces cerevisiae (S. cerevisiae), Yarrowia lipolytica, Pichia pastoris, or Kluyveromyces marxianus. In some embodiments, the genetically modified cell (e.g., microbe) is a yeast that is not Saccharomyces cerevisiae (S. cerevisiae).

[0105] In some embodiments, the trans gene that encodes a ferredoxin protein is integrated into the genome of the genetically modified cell (e.g., microbe) (e.g., S. cerevisiae or E. coli). The transgene may be integrated within an expression cassette that appropriately drives expression of the ferredoxin protein. For those embodiments in which genome integration of the transgene is preferred or desired, known methods of integration can be used, including but not limited to, Cas-based systems (e.g., Cas9, Casl2, etc.), homologous recombination, gene gun, conjugation protocols, lambda red, etc. The transgene encoding a ferredoxin may be integrated into a transcriptionally active genomic locus of the genetically modified microbe. In some embodiments, the genetically modified cell (e.g., microbe) is a yeast, and the transgene is integrated into the autonomously replicating sequence 1021 locus (ARS1021). Alternatively, in some embodiments, the transgene may not be integrated into the genome, and instead may express the ferredoxin protein from, for example, a vector (e.g., a plasmid).

[0106] An expression cassette or vector for expressing the transgene encoding a ferredoxin protein comprises an open reading frame encoding a ferredoxin protein described herein. In some embodiments, the expression cassette or vector includes regulatory elements necessary for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a terminator. In addition, enhancers may be included. These elements may be operably linked to a sequence that encodes ferredoxin protein described herein. Suitable promoters that can be used may include but are not limited to GALI, TEF2, TEF1, TDH3, ENO2, pCCW12, EF-la promoter, CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. In some embodiments, the promoter is pCCW12. In some embodiments, an inducible or repressible promoter, such as GALI, GAL2, GAL7, GAL 10, CUP1, MET3, MET17, or MET25, may be used. Inducible promoters operably link the expression of a target gene (e.g., the nucleic acid sequence encoding a ferredoxin protein) to a specific signal or a particular biotic or abiotic factor. Types of inducible promoters that may be utilized in the disclosed vectors include, but are not limited to, chemically-inducible promoters (i.e., antibiotics, steroids, metals, etc.), light-inducible promoters, heat-inducible promoters, and hypoxia-inducible promoters. Transcription terminators that may be used are also known in the art (see Bittner et al. (1987) Methods Enzymol 153: 516-544), and include but are not limited to, tYOL036W, GAT2, Rho-dependent terminators, Rho-independent terminators, poly-A sequences, and the like. In some embodiments, the terminator is tYOL036W.

[0107] In some embodiments, the genetically modified cell (e.g., microbe) may comprise at least one additional genetic modification besides the transgene encoding the ferredoxin protein.

[0108] Bioproduction of carotenoids may rely on a cell (e.g., microbe) that expresses a heterologous carotenoid biosynthetic enzyme. In some embodiments, the carotenoid biosynthetic enzymes include but are not limited to, e.g., geranylgeranyl diphosphate synthase (e.g., CrtE), bifunctional lycopene cyclase / phytoene synthase (e.g., CrtYB, CarRA, or CarRP), phytoene desaturase (e.g., Crtl or CarB), P-carotene hydroxylase (e.g., CrtZ or CHYb), and P-carotene ketolase (e.g., CrtW). In some embodiments, the CrtZ gene is not SsCrtZ. A cell (e.g., microbe) described herein may or may not endogenously express a carotenoid biosynthetic enzyme. In some embodiments, the genetically modified cell (e.g., microbe) described herein may comprise a nucleic acid (e.g., a transgene) encoding a carotenoid biosynthetic enzyme. In some embodiments, the genetically modified cell (e.g., microbe) described herein may comprise a nucleic acid (e.g., a transgene) encoding one or more carotenoid biosynthetic enzymes selected from geranylgeranyl diphosphate synthase (e.g., CrtE), bifunctional lycopene cyclase / phytoene synthase (e.g., CrtYB, CarRA, or CarRP), phytoene desaturase (e.g., Crtl or CarB), P-carotene hydroxylase (e.g., CrtZ or CHYb), and P-carotene ketolase (e.g., CrtW). In some embodiments, the genetically modified cell (e.g., microbe) described herein may comprise a nucleic acid (e.g., a transgene) encoding CrtE, CarRP, CarB, and / or CrtZ. In some embodiments, the CrtZ gene is not SsCrtZ.

[0109] In some aspects, provided herein is a genetically modified cell (e.g., microbe) comprising (i) a nucleic acid (e.g., a transgene) encoding a carotenoid biosynthetic enzyme disclosed herein, and (ii) a transgene that encodes a ferredoxin protein disclosed herein. In some embodiments, the genetically modified cell (e.g., microbe) comprises (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a transgene that encodes a native ferredoxin protein, wherein the transgene enhances the production of the carotenoid.

[0110] Bioproduction of a carotenoid can be enabled or increased by a genetically modified microbe cell (e.g., microbe) described herein. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing a carotenoid, e.g., zeaxanthin or a derivative thereof. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing one or more carotenoids selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and betacryptoxanthin. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing zeaxanthin and / or astaxanthin. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure does not produce violaxanthin.

[0111] In some embodiments, a genetically modified cell (e.g., microbe) of the present disclosure may produce at least about 0.1 pg / L - e.g., at least about 0.2 pg / L, at least about 0.3 pg / L, at least about 0.4 pg / L, at least about 0.5 pg / L, at least about 0.6 pg / L, at least about 0.7 pg / L, at least about 0.8 pg / L, at least about 0.9 pg / L, at least about 1.0 pg / L, at least about 1.1 pg / L, at least about 1.2 pg / L, at least about 1.3 pg / L, at least about 1.4 pg / L, at least about 1.5 pg / L, at least about 1.6 pg / L, at least about 1.7 pg / L, at least about 1.8 pg / L, at least about 1.9 pg / L, at least about 2.0 pg / L, at least about 2.1 pg / L, at least about 2.2 pg / L, at least about 2.3 pg / L, at least about 2.4 pg / L, at least about 2.5 pg / L, at least about 3.0 pg / L, at least about 4.0 pg / L, at least about 5.0 pg / L, at least about 10.0 pg / L, at least about 15.0 pg / L, at least about 20.0 pg / L, at least about 25.0 pg / L, at least about 30.0 pg / L, at least about 35.0 pg / L, at least about 40.0 pg / L, at least about 45.0 pg / L, at least about 50.0 pg / L, at least 100.0 pg / L, at least about 150.0 pg / L, at least about 200.0 pg / L, at least about 250.0 pg / L, at least about 300.0 pg / L, at least about 350.0 pg / L, at least about 400.0 pg / L, at least about 450.0 pg / L, at least about 500.0 pg / L, at least about 600.0 pg / L, at least about 700.0 pg / L, at least about 800.0 pg / L, at least about 900.0 pg / L, at least about 1.00 mg / L, at least about 1.25 mg / L, at least about 1.50 mg / L, at least about 1.75 mg / L, at least about 2.00 mg / L, at least about 2.25 mg / L, at least about 2.50 mg / L, at least about 2.75 mg / L, at least about 3.00 mg / L, at least about 3.25 mg / L, at least about 3.50 mg / L, at least about 3.75 mg / L, at least about 4.00 mg / L, at least about 4.00 mg / L, at least about 4.25 mg / L, at least about 4.50 mg / L, at least about 4.75 mg / L, at least about 5.00 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about 10 mg / L, at least about 15 mg / L, at least about 20 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 35 mg / L, at least about 40 mg / L, at least about 45 mg / L, at least about 50 mg / L, at least about 55 mg / L, at least about 60 mg / L, at least about 65 mg / L, at least about 70 mg / L, at least about 75 mg / L, at least about 80 mg / L, at least about 85 mg / L, at least about 90 mg / L, at least about 95 mg / L, at least about 100 mg / L, at least about 105 mg / L, at least about 110 mg / L, at least about 115 mg / L, at least about 120 mg / L, at least about 125 mg / L, at least about 130 mg / L, at least about 135 mg / L, at least about 140 mg / L, at least about 145 mg / L, at least about 150 mg / L, at least about 155 mg / L, at least about 160 mg / L, at least about 165 mg / L, at least about 170 mg / L, at least about 175 mg / L, at least about 180 mg / L, at least about 185 mg / L, at least about 190 mg / L, at least about 195 mg / L, or at least about 200 mg / L or more of a carotenoid (e.g., zeaxanthin or a derivative thereof) within about 48 hours of culture. Longer or shorter periods of culture time are also contemplated.

[0112] In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing an increased level of a carotenoid (e.g., zeaxanthin or a derivative thereof), compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the ferredoxin protein. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing an increased level of a carotenoid selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin, compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the ferredoxin protein. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing an increased level of zeaxanthin and / or astaxanthin, compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the ferredoxin protein. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure does not produce an increased level (i.e., produces similar or a lower level) of violaxanthin, compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the ferredoxin protein.

[0113] In some embodiments, a genetically modified cell (e.g., microbe) of the present disclosure may produce at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2- fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, or at least 10-fold of a carotenoid (e.g., zeaxanthin or a derivative thereof), compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the ferredoxin protein.

[0114] In some embodiments, a genetically modified cell (e.g., microbe) of the present disclosure may produce at least 1.1-fold, at least 1.2- fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2- fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, or at least 10-fold of a carotenoid selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin, compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the ferredoxin protein.

[0115] In some embodiments, a genetically modified cell (e.g., microbe) of the present disclosure may produce at least 1.1-fold, at least 1.2- fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2- fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, or at least 10-fold of zeaxanthin and / or astaxanthin, compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the ferredoxin protein.

[0116] In some embodiments, the genetically modified cell (e.g., microbe) is capable of producing, or producing at an increased level of a carotenoid (e.g., zeaxanthin or a derivative thereof) when cultured in a culture medium comprising an iron source. In some embodiments, the iron source is iron sulfate, e.g., iron sulfate heptahydrate.

[0117] In some embodiments, provided herein is a genetically modified cell comprising a transgene that encodes a native ferredoxin protein, wherein the genetically modified cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the native ferredoxin protein. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0118] In some embodiments, provided herein is a genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the ferredoxin protein, and wherein the carotenoid is not violaxanthin. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0119] In some embodiments, provided herein is a genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the cell is capable of producing an increased level of zeaxanthin, or a derivative thereof, compared to a cell having the same genetic background but without the transgene encoding the ferredoxin protein, and wherein the derivative of zeaxanthin is not violaxanthin. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0120] In some embodiments, provided herein is a genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the ferredoxin protein, and wherein the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0121] In some embodiments, provided herein is a genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the ferredoxin protein, and wherein the carotenoid is zeaxanthin or astaxanthin. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0122] In some aspects, provided herein is a genetically modified cell (e.g., microbe) comprising a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP. In some embodiments, the genetically modified cell (e.g., microbe) provided herein expresses the CBP. In some embodiments, the genetically modified cell (e.g., microbe) provided herein comprises a genetic modification capable of increasing the expression of a CBP.

[0123] CBPs are carrier proteins that, among their other functions, bind to a carotenoid and facilitate the intracellular transport and / or storage of the carotenoid. In some embodiments, the CBP disclosed herein binds specifically to xanthophylls. In some embodiments, the CBP disclosed herein binds specifically to zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof. In some embodiments, the CBP disclosed herein binds specifically to zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and / or beta-cryptoxanthin. In some embodiments, the CBP disclosed herein binds specifically to zeaxanthin and / or astaxanthin. In some embodiments, the CBP disclosed herein does not bind to carotenes. In some embodiments, the CBP disclosed herein does not bind to lycopene, a-carotene, P- carotene, and / or / -carotene. In some embodiments, the CBP disclosed herein does not bind to P-carotene.

[0124] The genetically modified cell (e.g., microbe) described herein may or may not express an endogenous CBP. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene that encodes a wild-type CBP or a genetic modification capable of increasing the expression of a wild-type CBP. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene that encodes an engineered CBP or a genetic modification capable of increasing the expression of an engineered CBP. The engineered CBP may be a functionally active fragment of a wild-type CBP, a CBP fused with a tag (e.g., a peptide tag, such as a Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, or V5 tag) or a reporter protein (e.g., a GFP protein, a luciferase protein, etc.), and / or a mutated CBP with one or more amino acid deletions, insertions, and / or substitutions. In preferred embodiments, the mutated CBP maintains or increases its activity in mediating carotenoid transport and / or storage.

[0125] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP that is native to the cell (e.g., microbe), i.e., from the same species as the cell (e.g., microbe), or a genetic modification capable of increasing the expression of a CBP that is native to the cell (e.g., microbe). In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP that is heterologous to the cell (e.g., microbe), i.e., from a different species as the cell (e.g., microbe), or a genetic modification capable of increasing the expression of a CBP that is heterologous to the cell (e.g., microbe). In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a bacterial CBP (e.g., orange carotenoid protein), or a genetic modification capable of increasing the expression of a bacterial CBP. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a plant CBP (e.g., light-harvesting chlorophyll a / b-binding proteins), or a genetic modification capable of increasing the expression of a plant CBP. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP comprising a START domain or a START-like domain, or a genetic modification capable of increasing the expression of a CBP comprising a START domain or a START-like domain. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP selected from the steroidogenic acute regulatory lipid transfer (START) protein family or the Aster family of proteins, or a genetic modification capable of increasing the expression of a CBP selected from the START protein family or the Aster family of proteins. In some embodiments, the CBP selected from the START protein family is Bombyx mori CBP or a Bombyx mori CBP ortholog. In some embodiments, the CBP selected from the START protein family is Bombyx mori CBP. In some embodiments, the CBP selected from the Aster family of proteins is Aster-A, Aster -B, and Aster -C, or homologs thereof. In some embodiments, the CBP selected from the Aster family of proteins is human Aster-A (Hs Aster A), or a human Aster-A homolog.

[0126] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP that is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof, or comprises a genetic modification capable of increasing the expression of a CBP that is not hSapB, SPF, HgCRA2, or HsApoB, or any variant thereof.

[0127] Exemplary nucleic acid and protein sequences of the CBP encompassed within the scope of compositions-of-matter and methods of the present disclosure are shown in Table 2 below.

[0128] Table 2. Exemplary DNA and Amino Acid Sequences of CBP

[0129] As is well-known to those skilled in the art, polypeptides having substantial sequence similarities can have the same or similar catalytic and / or functional activity. Accordingly, in some embodiments, a derivative, equivalent, variant, fragment, or mutant of a CBP described herein or fragment thereof may also be suitable for the methods and compositions provided herein.

[0130] There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code. An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed. Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0131] For a CBP amino acid sequence, corresponding nucleotide sequences that can encode the CBP can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Therefore, description and / or disclosure of a CBP amino acid sequence herein should be considered to also include description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0132] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP, wherein the CBP may comprise, or consist of, the amino acid sequence of SEQ ID NO: 12. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP, wherein the CBP has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 12.

[0133] In some embodiments, the CBP has an amino acid sequence that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, or 297 consecutive amino acids of SEQ ID NO: 12.

[0134] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP, wherein the CBP comprises an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 12.

[0135] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP, wherein the CBP may comprise, or consist of, the amino acid sequence of SEQ ID NO: 14. In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP, wherein the CBP has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 14.

[0136] In some embodiments, the CBP has an amino acid sequence that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 205, 210, 215, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, or 230 consecutive amino acids of SEQ ID NO: 14.

[0137] In some embodiments, the genetically modified cell (e.g., microbe) comprises a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP, wherein the CBP comprises an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 14.

[0138] In some embodiments, provided herein is a genetically modified cell (e.g., microbe) comprising a transgene that encodes a CBP, and the transgene encoding the CBP may comprise, or consist of, the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the transgene encoding a CBP as described herein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 11.

[0139] In some embodiments, provided herein is a genetically modified cell (e.g., microbe) comprising a transgene that encodes a CBP, and the transgene encoding the CBP may comprise, or consist of, the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the transgene encoding a CBP as described herein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 13.

[0140] For the purposes of the present disclosure, any of the foregoing CBPs can be expressed in a host cell, transgenic cell or modified cell (e.g., a host microbe, transgenic microbe, or modified microbe), and any of the foregoing nucleic acids may be incorporated into a host cell, transgenic cell or modified cell (e.g., a host microbe, transgenic microbe, or modified microbe) in order to produce carotenoids according to the disclosed methods.

[0141] The genetically modified cell that comprises a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP may be a prokaryote or a eukaryote. For example, the genetically modified cell may be a microbe. The genetically modified cell (e.g., microbe) may be a bacterium, an archaeon, a protozoon, an alga, or a fungus.

[0142] In some embodiments, the genetically modified cell (e.g., microbe) is a prokaryote. Model prokaryotic systems that may be utilized as a genetically modified cell (e.g., microbe) include but are not limited to Escherichia coli (E. coli), an Acinetobacter species, a Pseudomonas species, a Streptomyces species, and a Mycobacterium species. Additional suitable prokaryotic expression systems include, but are not limited to, Klebsiella, Lactococcus, Mannheimia, Corynebacterium, Vibrio, and Bacillis. In some embodiments, the genetically modified cell (e.g., microbe) is a bacterium, e.g., Escherichia coli (E. coli).

[0143] In some embodiments, the genetically modified cell (e.g., microbe) is a eukaryote. Model eukaryotic systems that may be utilized as a genetically modified cell (e.g., microbe) include, but are not limited to, Saccharomyces cerevisiae (S. cerevisiae) or other yeast species (e.g., Yarrowia lipolytica, Pichia pastoris, or Kluyveromyces marxianus),' a filamentous fungus, optionally selected from an Aspergillus species and a Trichoderma species; an alga, optioncdly selected from Dunaliella salina, Haematococcus lacustris, Botryococcus braunii, Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp., and Tetraselmis suecia; and an amoeba, which is optionally Diciyosielium discoideum. Additional suitable eukaryotic expression systems include, but are not limited to, Pichia pastoris, Rhodosporidium toruloides, Aspergillus (oryzae, nidulans, or niger), Trichoderma reesei, and Penicillium chrysogenum, etc. In some embodiments, the genetically modified cell (e.g., microbe) is a yeast, e.g., Saccharomyces cerevisiae (S. cerevisiae), Yarrowia lipolytica, Pichia pastoris, or Kluyveromyces marxianus. In some embodiments, the genetically modified cell (e.g., microbe) is a yeast that is not Saccharomyces cerevisiae (S. cerevisiae).

[0144] In some embodiments, the trans gene that encodes a CBP is integrated into the genome of the genetically modified cell (e.g., microbe) (e.g., S. cerevisiae or E. coli). The transgene may be integrated within an expression cassette that appropriately drives expression of the CBP. For those embodiments in which genome integration of the transgene is preferred or desired, known methods of integration can be used, including but not limited to, Cas-based systems (e.g., Cas9, Casl2, etc.), homologous recombination, gene gun, conjugation protocols, lambda red, etc. The transgene encoding a CBP may be integrated into a transcriptionally active genomic locus of the genetically modified microbe. In some embodiments, the genetically modified cell (e.g., microbe) is a yeast, and the transgene is integrated into the autonomously replicating sequence 511 locus (ARS511). Alternatively, in some embodiments, the transgene may not be integrated into the genome, and instead may express the CBP from, for example, a vector (e.g., a plasmid).

[0145] An expression cassette or vector for expressing the transgene encoding a CBP comprises an open reading frame encoding a CBP described herein. In some embodiments, the expression cassette or vector includes regulatory elements necessary for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a terminator. In addition, enhancers may be included. These elements may be operably linked to a sequence that encodes CBP described herein. Suitable promoters that can be used may include but are not limited to GALI, TEF2, TEF1, TDH3, ENO2, pGAL7, EF- la promoter, CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. In some embodiments, the promoter is pGAL7. In some embodiments, an inducible or repressible promoter, such as GALI, GAL2, GAL7, GAL10, CUP1, MET3, MET17, or MET25, may be used. Inducible promoters operably link the expression of a target gene (e.g., the nucleic acid sequence encoding a CBP) to a specific signal or a particular biotic or abiotic factor. Types of inducible promoters that may be utilized in the disclosed vectors include, but are not limited to, chemically-inducible promoters ( / .<?., antibiotics, steroids, metals, etc.), light-inducible promoters, heat-inducible promoters, and hypoxia-inducible promoters. Transcription terminators that may be used are also known in the art (see Bittner et al. (1987) Methods Enzymol 153: 516-544), and include but are not limited to, tHSP26, GAT2, Rho-dependent terminators, Rho-independent terminators, poly-A sequences, and the like. In some embodiments, the terminator is tHSP26.

[0146] In some embodiments, the genetically modified cell (e.g., microbe) may comprise at least one additional genetic modification besides the transgene encoding the CBP or the genetic modification capable of increasing the expression of the CBP.

[0147] Bioproduction of carotenoids may rely on a cell (e.g., microbe) that expresses a heterologous carotenoid biosynthetic enzyme. In some embodiments, the carotenoid biosynthetic enzymes include but are not limited to, e.g., geranylgeranyl diphosphate synthase (e.g., CrtE), bifunctional lycopene cyclase / phytoene synthase (e.g., CrtYB, CarRA, or CarRP), phytoene desaturase (e.g., Crtl or CarB), P-carotene hydroxylase (e.g., CrtZ or CHYb), and P-carotene ketolase (e.g., CrtW). A cell (e.g., microbe) described herein may or may not endogenously express a carotenoid biosynthetic enzyme. In some embodiments, the genetically modified cell (e.g., microbe) described herein may comprise a nucleic acid (e.g., a transgene) encoding a carotenoid biosynthetic enzyme. In some embodiments, the genetically modified cell (e.g., microbe) described herein may comprise a nucleic acid (e.g., a transgene) encoding one or more carotenoid biosynthetic enzymes selected from geranylgeranyl diphosphate synthase (e.g., CrtE), bifunctional lycopene cyclase / phytoene synthase (e.g., CrtYB, CarRA, or CarRP), phytoene desaturase (e.g., Crtl or CarB), P-carotene hydroxylase (e.g., CrtZ or CHYb), and P-carotene ketolase (e.g., CrtW). In some embodiments, the genetically modified cell (e.g., microbe) described herein may comprise a nucleic acid (e.g., a transgene) encoding CrtE, CarRP, CarB, and / or CrtZ.

[0148] In some aspects, provided herein is a genetically modified cell (e.g., microbe) comprising (i) a nucleic acid (e.g., a transgene) encoding a carotenoid biosynthetic enzyme disclosed herein, and (ii) a transgene that encodes a CBP disclosed herein, wherein the transgene encoding the CBP enhances the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof. In some embodiments, the genetically modified cell (e.g., microbe) comprises (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a transgene that encodes a heterologous CBP disclosed herein, wherein the transgene encoding the CBP enhances the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof. In some aspects, provided herein is a genetically modified cell comprising (i) a nucleic acid (e.g., a transgene) encoding a carotenoid biosynthetic enzyme disclosed herein, and (ii) a genetic modification capable of increasing the expression of a CBP, wherein the genetic modification enhances the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof.

[0149] Bioproduction of a carotenoid can be enabled or increased by a genetically modified microbe cell (e.g., microbe) described herein. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing a carotenoid, e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing one or more carotenoids selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing zeaxanthin and / or astaxanthin.

[0150] In some embodiments, a genetically modified cell (e.g., microbe) of the present disclosure may produce at least about 0.1 pg / L - e.g., at least about 0.2 pg / L, at least about 0.3 pg / L, at least about 0.4 pg / L, at least about 0.5 pg / L, at least about 0.6 pg / L, at least about 0.7 pg / L, at least about 0.8 pg / L, at least about 0.9 pg / L, at least about 1.0 pg / L, at least about 1.1 |ig / L, at least about 1.2 pg / L, at least about 1.3 pg / L, at least about 1.4 pg / L, at least about 1.5 pg / L, at least about 1.6 pg / L, at least about 1.7 pg / L, at least about 1.8 pg / L, at least about 1.9 pg / L, at least about 2.0 pg / L, at least about 2.1 pg / L, at least about 2.2 pg / L, at least about 2.3 pg / L, at least about 2.4 pg / L, at least about 2.5 pg / L, at least about 3.0 pg / L, at least about 4.0 pg / L, at least about 5.0 pg / L, at least about 10.0 pg / L, at least about 15.0 pg / L, at least about 20.0 pg / L, at least about 25.0 pg / L, at least about 30.0 pg / L, at least about 35.0 pg / L, at least about 40.0 pg / L, at least about 45.0 pg / L, at least about 50.0 pg / L, at least 100.0 pg / L, at least about 150.0 pg / L, at least about 200.0 pg / L, at least about 250.0 pg / L, at least about 300.0 pg / L, at least about 350.0 pg / L, at least about 400.0 pg / L, at least about 450.0 pg / L, at least about 500.0 pg / L, at least about 600.0 pg / L, at least about 700.0 pg / L, at least about 800.0 pg / L, at least about 900.0 pg / L, at least about 1.00 mg / L, at least about 1.25 mg / L, at least about 1.50 mg / L, at least about 1.75 mg / L, at least about 2.00 mg / L, at least about 2.25 mg / L, at least about 2.50 mg / L, at least about 2.75 mg / L, at least about 3.00 mg / L, at least about 3.25 mg / L, at least about 3.50 mg / L, at least about 3.75 mg / L, at least about 4.00 mg / L, at least about 4.00 mg / L, at least about 4.25 mg / L, at least about 4.50 mg / L, at least about 4.75 mg / L, at least about 5 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about 10 mg / L, at least about 15 mg / L, at least about 20 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 35 mg / L, at least about 40 mg / L, at least about 45 mg / L, at least about 50 mg / L, at least about 55 mg / L, at least about 60 mg / L, at least about 65 mg / L, at least about 70 mg / L, at least about 75 mg / L, at least about 80 mg / L, at least about 85 mg / L, at least about 90 mg / L, at least about 95 mg / L, at least about 100 mg / L, at least about 105 mg / L, at least about 110 mg / L, at least about 115 mg / L, at least about 120 mg / L, at least about 125 mg / L, at least about 130 mg / L, at least about 135 mg / L, at least about 140 mg / L, at least about 145 mg / L, or at least about 150 mg / L or more, of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof) within about 48 hours of culture. Longer or shorter periods of culture time are also contemplated.

[0151] In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing an increased level of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof), compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the CBP or the genetic modification capable of increasing the expression of the CBP. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing an increased level of a carotenoid selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin, compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the CBP or the genetic modification capable of increasing the expression of the CBP. In some embodiments, the genetically modified cell (e.g., microbe) of the present disclosure is capable of producing an increased level of zeaxanthin and / or astaxanthin, compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the CBP or the genetic modification capable of increasing the expression of the CBP.

[0152] In some embodiments, a genetically modified cell (e.g., microbe) of the present disclosure may produce at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof), compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the CBP or the genetic modification capable of increasing the expression of the CBP.

[0153] In some embodiments, a genetically modified cell (e.g., microbe) of the present disclosure may produce at least 1.1-fold, at least 1.2- fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold of a carotenoid selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin, compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the CBP or the genetic modification capable of increasing the expression of the CBP.

[0154] In some embodiments, a genetically modified cell (e.g., microbe) of the present disclosure may produce at least 1.1-fold, at least 1.2- fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2- fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15 -fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold of zeaxanthin and / or astaxanthin, compared to a cell (e.g., microbe) having the same genetic background but without the transgene encoding the CBP or the genetic modification capable of increasing the expression of the CBP.

[0155] In some embodiments, the genetically modified cell (e.g., microbe) is capable of producing, or producing at an increased level of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof) when cultured in a culture medium comprising an iron source. In some embodiments, the iron source is iron sulfate, e.g., iron sulfate heptahydrate.

[0156] In some embodiments, provided herein is a genetically modified cell comprising a transgene that encodes a heterologous CBP, wherein the genetically modified cell is capable of producing an increased level of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof) compared to a cell having the same genetic background but without the transgene encoding the heterologous CBP. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0157] In some embodiments, provided herein is a genetically modified cell comprising a transgene that encodes a heterologous CBP, wherein the cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the heterologous CBP, and wherein the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0158] In some embodiments, provided herein is a genetically modified cell comprising a transgene that encodes a heterologous CBP, wherein the cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the heterologous CBP, and wherein the carotenoid is zeaxanthin or astaxanthin. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0159] In some embodiments, provided herein is a genetically modified cell comprising a transgene that encodes a heterologous CBP, wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0160] Methods of bioproduction of carotenoids The present disclosure provides compositions and / or methods for producing a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof). In some embodiments, the compositions and / or methods disclosed herein can be used to produce a xanthophyll. In some embodiments, the compositions and / or methods disclosed herein can be used to produce P-carotene. In some embodiments, the compositions and / or methods disclosed herein can be used to produce zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof. In some embodiments, the compositions and / or methods disclosed herein can be used to produce zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and / or -cryptoxanthin. In some embodiments, the compositions and / or methods disclosed herein can be used to produce zeaxanthin and / or astaxanthin.

[0161] In some embodiments, the present disclosure provides methods of producing a carotenoid (e.g., zeaxanthin or a derivative thereof), comprising culturing a genetically modified cell (e.g., a microbe) disclosed herein in a culture medium. In some embodiments, the present disclosure provides methods of producing zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and / or P-cryptoxanthin, comprising culturing a genetically modified cell (e.g., a microbe) disclosed herein in a culture medium. In some embodiments, the present disclosure provides methods of producing zeaxanthin and / or astaxanthin, comprising culturing a genetically modified cell (e.g., a microbe) disclosed herein in a culture medium. In some embodiments, the present disclosure provides methods of producing a carotenoid comprising culturing a genetically modified cell (e.g., a microbe) disclosed herein in a culture medium, and the carotenoid is not violaxanthin.

[0162] In some embodiments, the present disclosure provides a method of producing a carotenoid (e.g., zeaxanthin or a derivative thereof), comprising culturing the genetically modified cell (e.g., a microbe) that comprise a transgene encoding a ferredoxin protein in a culture medium, wherein the ferredoxin protein is not YAH1 or ferredoxin-3.

[0163] In some embodiments, the present disclosure provides a method of producing a carotenoid (e.g., zeaxanthin or a derivative thereof), comprising culturing the genetically modified cell (e.g., a microbe) that comprise a transgene encoding a ferredoxin protein selected from APD1, ATM1, ILV3, and NAR1 in a culture medium.

[0164] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as .S', cerevisiae or E. coli) comprising a transgene that encodes a ferredoxin protein that may comprise, or consist of, SEQ ID NO: 2. In some embodiments, the ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 2. In some embodiments, the ferredoxin protein can comprise an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 2.

[0165] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as .S', cerevisiae or E. coli) comprising a transgene that encodes a ferredoxin protein that may comprise, or consist of, SEQ ID NO: 4. In some embodiments, the ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 4. In some embodiments, the ferredoxin protein can comprise an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 4.

[0166] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as S. cerevisiae or E. coli) comprising a transgene that encodes a ferredoxin protein that may comprise, or consist of, SEQ ID NO: 6. In some embodiments, the ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 6. In some embodiments, the ferredoxin protein can comprise an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 6.

[0167] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as .S'. cerevisiae or E. coli) comprising a transgene that encodes a ferredoxin protein that may comprise, or consist of, SEQ ID NO: 8. In some embodiments, the ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 8. In some embodiments, the ferredoxin protein can comprise an amino acid sequence comprising at least one (e.g., 1 , 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 8.

[0168] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as .S', cerevisiae or E. coll) comprising a transgene that encodes a ferredoxin protein that may comprise, or consist of, SEQ ID NO: 10. In some embodiments, the ferredoxin protein as described herein at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 10. In some embodiments, the ferredoxin protein can comprise an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 10.

[0169] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as S. cerevisiae or E. coli) comprising a transgene that may comprise, or consist of, SEQ ID NO: 1. In some embodiments, the transgene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with the sequence of SEQ ID NO: 1.

[0170] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as S. cerevisiae or E. coli) comprising a transgene that may comprise, or consist of, SEQ ID NO: 3. In some embodiments, the transgene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 3.

[0171] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as .S'. cerevisiae or E. coli) comprising a transgene that may comprise, or consist of, SEQ ID NO: 5. In some embodiments, the transgene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 5.

[0172] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as S. cerevisiae or E. coli) comprising a transgene that may comprise, or consist of, SEQ ID NO: 7. In some embodiments, the transgene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 7.

[0173] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as S. cerevisiae or E. coli) comprising a transgene that may comprise, or consist of, SEQ ID NO: 9. In some embodiments, the transgene encoding a ferredoxin protein as described herein has at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 9.

[0174] Various prokaryotic and eukaryotic expression systems can be utilized for the disclosed methods. In some embodiments, the genetically modified cell (e.g., microbe) used in the methods may be a prokaryote, including but not limited to Escherichia coli (E coli). an Acinetobacter species, a Pseudomonas species, a Streptomyces species, and a Mycobacterium species. Additionally suitable prokaryotic expression systems include, but are not limited to, Klebsiella, Lactococcus, Mannheimia, Corynebacterium, Vibrio, and Bacillis. In some embodiments, the genetically modified cell (e.g., microbe) used in the methods may be a eukaryote, including but not limited to Saccharomyces cerevisiae (S. cerevisiae) or other yeast species (e.g., Yarrowia lipolytica, Pichia pastoris, or Kluyveromyces marxianusy, a filamentous fungi, optionally selected from an Aspergillus species and a Trichoderma species; an algae, optionally selected from Dunaliella salina, Elaematococcus lacustris, Botryococcus braunii, Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp., and Tetraselmis suecia', and an amoeba, which is optionally Dictyostelium discoideum. Additional suitable eukaryotic expression systems include, but are not limited to, Pichia pastoris, Rhodosporidium tondoides, Aspergillus (oryzae, nidulans, niger), Trichoderma reesei, and Penicillium chrysogenum.

[0175] The disclosed methods can be carried out in a bioproduction reactor, fermentation tank, culture flask, or other suitable containers for bioproduction. In some embodiments, the methods described herein comprise culturing the genetically modified cell (e.g., microbe) in a culture medium in a batch fermentation, such as the batch fermentation exemplified in Example 2 below. Various different culture mediums can be selected based on the particular transgenic species used and the growth conditions, among other things. In some embodiments, a defined culture medium is used. In some embodiments, minimal culture medium may be supplemented as needed to optimize growth and / or carotenoid production of a given genetically modified microbial cell type. For example, in some embodiments, such as those utilizing genetically modified .S', cerevisiae, the culture medium may comprise about l%-5% (e.g., 3%) w / v maltodextrin, about 0.05%-0.5% (e.g., 0.2%) w / v glucose, alphaamylase, or any combination thereof. In some embodiments, the culture medium may comprise yeast extract, e.g., 0.5-5 g / L (e.g., 2 g / L) of yeast extract, and / or an iron source. In some embodiments, the iron source is iron sulfate, e.g., iron sulfate heptahydrate. The iron sulfate may be at a concentration from 0. 1 mM to 20 mM, from 0.5 mM to 10 mM, or from 1.0 mM to 5 mM, e.g., about 0.5 mM, 1.0 mM, about 1.2 mM, about 1.5 mM, about 1.7 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.4 mM, about 4 mM, about 4.5 mM, or about 5 mM.

[0176] The disclosed methods may further comprise extracting the produced carotenoid from the genetically modified cell (e.g., microbe) . Various extraction methods known in the art can be used to extract carotenoid from the genetically modified cell (e.g., microbe) , depending on the cell type. The extraction step may comprise permeabilizing the cell and / or solubilizing the produced carotenoids. Exemplary methods to extract carotenoids from yeast (e.g., .S', cerevisiae) are shown in Example 1-10 below.

[0177] The disclosed methods of bioproduction may be further optimized and developed to increase yield. For example, in some embodiments, the disclosed methods may produce at least about 0.1 pg / L, at least about 0.2 pg / L, at least about 0.3 pg / L, at least about 0.4 pg / L, at least about 0.5 pg / L, at least about 0.6 pg / L, at least about 0.7 pg / L, at least about 0.8 pg / L, at least about 0.9 pg / L, at least about 1.0 pg / L, at least about 1.1 pg / L, at least about 1.2 pg / L, at least about 1.3 pg / L, at least about 1.4 pg / L, at least about 1.5 pg / L, at least about 1.6 pg / L, at least about 1.7 pg / L, at least about 1.8 pg / L, at least about 1.9 pg / L, at least about 2.0 pg / L, at least about 2.1 pg / L, at least about 2.2 pg / L, at least about 2.3 pg / L, at least about 2.4 pg / L, at least about 2.5 pg / L, at least about 3.0 pg / L, at least about 3.5 pg / L, at least about 4.0 pg / L, at least about 4.5 pg / L, at least about 5.0 pg / L, at least about 5.5 pg / L, at least about 6.0 pg / L, at least about 6.5 pg / L, at least about 7.0 pg / L, at least about 7.5 pg / L, at least about 8.0 pg / L, at least about 8.5 pg / L, at least about 9.0 pg / L, at least about 9.5 pg / L, at least about 10.0 pg / L, at least about 20 pg / L, at least about 30 pg / L, at least about 40 pg / L, at least about 50 pg / L, at least about 75 pg / L, at least about 100 pg / L, at least about 150.0 pg / L, at least about 200.0 pg / L, at least about 250.0 pg / L, at least about 300.0 pg / L, at least about 350.0 pg / L, at least about 400.0 pg / L, at least about 450.0 pg / L, at least about 500.0 pg / L, at least about 600.0 pg / L, at least about 700.0 pg / L, at least about 800.0 pg / L, at least about 900.0 pg / L, at least about 1.00 mg / L, at least about 1.25 mg / L, at least about 1.50 mg / L, at least about 1.75 mg / L, at least about 2.00 mg / L, at least about 2.25 mg / L, at least about 2.50 mg / L, at least about 2.75 mg / L, at least about 3.00 mg / L, at least about 3.25 mg / L, at least about 3.50 mg / L, at least about 3.75 mg / L, at least about 4.00 mg / L, at least about 4.00 mg / L, at least about 4.25 mg / L, at least about 4.50 mg / L, at least about 4.75 mg / L, at least about 5.00 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about 10 mg / L, at least about 15 mg / L, at least about 20 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 35 mg / L, at least about 40 mg / L, at least about 45 mg / L, at least about 50 mg / L, at least about 55 mg / L, at least about 60 mg / L, at least about 65 mg / L, at least about 70 mg / L, at least about 75 mg / L, at least about 80 mg / L, at least about 85 mg / L, at least about 90 mg / L, at least about 95 mg / L, at least about 100 mg / L, at least about 105 mg / L, at least about 110 mg / L, at least about 115 mg / L, at least about 120 mg / L, at least about 125 mg / L, at least about 130 mg / L, at least about 135 mg / L, at least about 140 mg / L, at least about 145 mg / L, at least about 150 mg / L, at least about 155 mg / L, at least about 160 mg / L, at least about 165 mg / L, at least about 170 mg / L, at least about 175 mg / L, at least about 180 mg / L, at least about 185 mg / L, at least about 190 mg / L, at least about 195 mg / L, or at least about 200 mg / L or more of a carotenoid (e.g., zeaxanthin or a derivative thereof) within at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 36 hours, or at least about 48 hours of culture.

[0178] In some embodiments, the disclosed methods may produce at least about 0. 1 pg / L, at least about 0.2 pg / L, at least about 0.3 pg / L, at least about 0.4 pg / L, at least about 0.5 pg / L, at least about 0.6 pg / L, at least about 0.7 pg / L, at least about 0.8 pg / L, at least about 0.9 pg / L, at least about 1.0 pg / L, at least about 1.1 pg / L, at least about 1.2 pg / L, at least about 1.3 pg / L, at least about 1.4 pg / L, at least about 1.5 pg / L, at least about 1.6 pg / L, at least about 1.7 pg / L, at least about 1.8 pg / L, at least about 1.9 pg / L, at least about 2.0 pg / L, at least about 2.1 pg / L, at least about 2.2 pg / L, at least about 2.3 pg / L, at least about 2.4 pg / L, at least about 2.5 pg / L, at least about 3.0 pg / L, at least about 3.5 pg / L, at least about 4.0 pg / L, at least about 4.5 pg / L, at least about 5.0 pg / L, at least about 5.5 pg / L, at least about 6.0 pg / L, at least about 6.5 pg / L, at least about 7.0 pg / L, at least about 7.5 pg / L, at least about 8.0 pg / L, at least about 8.5 pg / L, at least about 9.0 pg / L, at least about 9.5 pg / L, at least about 10.0 pg / L, at least about 20 pg / L, at least about 30 pg / L, at least about 40 pg / L, at least about 50 pg / L, at least about 75 pg / L, at least about 100 pg / L, at least about 150.0 pg / L, at least about 200.0 pg / L, at least about 250.0 pg / L, at least about 300.0 pg / L, at least about 350.0 pg / L, at least about 400.0 pg / L, at least about 450.0 pg / L, at least about 500.0 pg / L, at least about 600.0 pg / L, at least about 700.0 pg / L, at least about 800.0 pg / L, at least about 900.0 pg / L, at least about 1.00 mg / L, at least about 1.25 mg / L, at least about 1.50 mg / L, at least about 1.75 mg / L, at least about 2.00 mg / L, at least about 2.25 mg / L, at least about 2.50 mg / L, at least about 2.75 mg / L, at least about 3.00 mg / L, at least about 3.25 mg / L, at least about 3.50 mg / L, at least about 3.75 mg / L, at least about 4.00 mg / L, at least about 4.00 mg / L, at least about 4.25 mg / L, at least about 4.50 mg / L, at least about 4.75 mg / L, at least about 5.00 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about 10 mg / L, at least about 15 mg / L, at least about 20 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 35 mg / L, at least about 40 mg / L, at least about 45 mg / L, at least about 50 mg / L, at least about 55 mg / L, at least about 60 mg / L, at least about 65 mg / L, at least about 70 mg / L, at least about 75 mg / L, at least about 80 mg / L, at least about 85 mg / L, at least about 90 mg / L, at least about 95 mg / L, at least about 100 mg / L, at least about 105 mg / L, at least about 110 mg / L, at least about 115 mg / L, at least about 120 mg / L, at least about 125 mg / L, at least about 130 mg / L, at least about 135 mg / L, at least about 140 mg / L, at least about 145 mg / L, at least about 150 mg / L, at least about 155 mg / L, at least about 160 mg / L, at least about 165 mg / L, at least about 170 mg / L, at least about 175 mg / L, at least about 180 mg / L, at least about 185 mg / L, at least about 190 mg / L, at least about 195 mg / L, or at least about 200 mg / L or more o! a carotenoid (e.g., zeaxanthin or a derivative thereof) within about 6 hours of culture or less, about 12 hours of culture or less, about 18 hours of culture or less, about 24 hours of culture or less, about 36 hours of culture or less, or about 48 hours of culture or less.

[0179] In some embodiments, the present disclosure provides a method of increasing production of a carotenoid (e.g., zeaxanthin or a derivative thereof), comprising culturing the genetically modified cell (e.g., microbe) described herein in a culture medium. In some embodiments, the present disclosure provides a method of increasing production of zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and / or -cryptoxanthin, comprising culturing a genetically modified cell (e.g., microbe) disclosed herein in a culture medium. In some embodiments, the present disclosure provides a method of increasing production of zeaxanthin and / or astaxanthin, comprising culturing a genetically modified cell (e.g., microbe) disclosed herein in a culture medium. In some embodiments, the present disclosure provides a method of increasing production of a carotenoid (e.g., zeaxanthin or a derivative thereof) comprising culturing a genetically modified cell (e.g., microbe) disclosed herein in a culture medium, and the carotenoid is not violaxanthin.

[0180] In some embodiments, the disclosed methods may produce an increased level of a carotenoid (e.g., zeaxanthin and / or astaxanthin) in a genetically modified cell (e.g., microbe) disclosed herein compared to methods of using a cell (e.g., microbe) having the same genetic background but without the trans gene encoding the ferredoxin protein. In some embodiments, the disclosed methods may produce at least 1. 1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of a carotenoid (e.g., zeaxanthin and / or astaxanthin) compared to methods of using a cell (e.g., microbe) having the same genetic background but without the transgene encoding the ferredoxin protein.

[0181] The present disclosure also provides methods of increasing a carotenoid production in a cell (e.g., microbe) , comprising contacting the cell (e.g., microbe) with an agent that increases gene copy number, expression, and / or activity of ferredoxin, wherein the ferredoxin is not YAH1 or ferredoxin- 3.

[0182] In some embodiments, provided herein is a method of increasing a carotenoid production in a cell (e.g., microbe) , comprising contacting the cell (e.g., microbe) with an agent that increases gene copy number, expression, and / or activity of ferredoxin, wherein the ferredoxin is selected from APD1, ATM1, ILV3, and NAR1.

[0183] In some embodiments, provided herein is a method of increasing production of a carotenoid in a cell (e.g., microbe) , wherein the carotenoid is not violaxanthin, comprising contacting the microbe with an agent that increases gene copy number, expression, and / or activity of ferredoxin.

[0184] In some embodiments, provided herein is a method of increasing production of a carotenoid selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin in a cell (e.g., microbe) , comprising contacting the cell (e.g., microbe) with an agent that increases gene copy number, expression, and / or activity of ferredoxin.

[0185] In some embodiments, provided herein is a method of increasing production of zeaxanthin and / or astaxanthin in a cell (e.g., microbe) , comprising contacting the cell (e.g., microbe) with an agent that increases gene copy number, expression, and / or activity of ferredoxin.

[0186] In some embodiments, the agent used in the methods disclosed herein is a nucleic acid (e.g., an expression vector encoding a ferredoxin protein), a protein, and / or a small molecule.

[0187] The present disclosure also provides batches of carotenoid produced by the methods disclosed herein. A bioproduction batch of carotenoid may have a chemical purity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or any values in between any of the two aforementioned values, and no single impurity of greater than 1%, no greater than about 0.5%, or greater than about 0.1%. The level of impurities in a given batch of carotenoid can be determined by high-performance liquid chromatography (HPLC) and other suitable techniques.

[0188] In some embodiments, the present disclosure provides methods of producing a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof), comprising culturing a genetically modified cell (e.g., a microbe) disclosed herein in a culture medium. In some embodiments, the present disclosure provides methods of producing zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and / or P-cryptoxanthin, comprising culturing a genetically modified cell (e.g., a microbe) disclosed herein in a culture medium. In some embodiments, the present disclosure provides methods of producing zeaxanthin and / or astaxanthin, comprising culturing a genetically modified cell (e.g., a microbe) disclosed herein in a culture medium.

[0189] In some embodiments, the present disclosure provides a method of producing a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof), comprising culturing the genetically modified cell (e.g., a microbe) that comprise a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP in a culture medium, wherein the CBP is selected from the steroidogenic acute regulatory lipid transfer (START) protein family or the Aster family of proteins.

[0190] In some embodiments, the present disclosure provides a method of producing a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof), comprising culturing the genetically modified cell (e.g., a microbe) that comprise a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP in a culture medium, wherein the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog, or human Aster-A (HsAsterA) or a human Aster-A homolog.

[0191] In some embodiments, the present disclosure provides a method of producing a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof), comprising culturing the genetically modified cell (e.g., a microbe) that comprise a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP in a culture medium, wherein the CBP is Bombyx mori CBP or human Aster-A (HsAsterA).

[0192] In some embodiments, the present disclosure provides a method of producing a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof), comprising culturing the genetically modified cell (e.g., a microbe) that comprise a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP in a culture medium, wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof. In some embodiments, the genetically modified cell is a genetically modified microbe.

[0193] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as .S', cerevisiae or E. coli) comprising a transgene that encodes a CBP that may comprise, or consist of, SEQ ID NO: 12. In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as S. cerevisiae or E. coli) comprising a genetic modification capable of increasing the expression of a CBP, wherein the CBP may comprise, or consist of, SEQ ID NO: 12. In some embodiments, the CBP as described herein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 12. In some embodiments, the CBP can comprise an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 12.

[0194] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as .S’, cerevisiae or E. coli) comprising a transgene that encodes a CBP that may comprise, or consist of, SEQ ID NO: 14. In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as S. cerevisiae or E. coli) comprising a genetic modification capable of increasing the expression of a CBP, wherein the CBP may comprise, or consist of, SEQ ID NO: 14. In some embodiments, the CBP as described herein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the sequence of SEQ ID NO: 14. In some embodiments, the CBP can comprise an amino acid sequence comprising at least one (e.g., 1, 2, 3, 4, or 5 or more) substitution mutation(s), addition(s), and / or deletion(s) relative to SEQ ID NO: 14.

[0195] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as .S', cerevisiae or E. coli) comprising a transgene that may comprise, or consist of, SEQ ID NO: 11. In some embodiments, the transgene encoding a CBP as described herein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with the sequence of SEQ ID NO: 11.

[0196] In some embodiments, the method comprises culturing a genetically modified cell (e.g., a microbe such as S. cerevisiae or E. coli) comprising a transgene that may comprise, or consist of, SEQ ID NO: 13. In some embodiments, the transgene encoding a CBP as described herein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with the sequence of SEQ ID NO: 13.

[0197] Various prokaryotic and eukaryotic expression systems can be utilized for the disclosed methods. In some embodiments, the genetically modified cell (e.g., microbe) used in the methods may be a prokaryote, including but not limited to Escherichia coli (E. coli), an Acinetobacter species, a Pseudomonas species, a Streptomyces species, and a Mycobacterium species. Additionally suitable prokaryotic expression systems include, but are not limited to, Klebsiella, Lactococcus, Mannheimia, Corynebacterium, Vibrio, and Bacillis. In some embodiments, the genetically modified cell (e.g., microbe) used in the methods may be a eukaryote, including but not limited to Saccharomyces cerevisiae (S. cerevisiae) or other yeast species (e.g., Yarrowia lipolytica, Pichia pastoris, or Kluyveromyces marxianus)-, a filamentous fungi, optionally selected from an Aspergillus species and a Trichoderma species; an algae, optionally selected from Dunaliella salina, Haematococcus lacustris, Botryococcus braunii, Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp., and Tetraselmis suecia', and an amoeba, which is optionally Dictyostelium discoideum. Additional suitable eukaryotic expression systems include, but are not limited to, Pichia pastoris, Rhodosporidium toruloides, Aspergillus (oryzae, nidulans, niger), Trichoderma reesei, and Penicillium chrysogenum.

[0198] The disclosed methods can be carried out in a bioproduction reactor, fermentation tank, culture flask, or other suitable containers for bioproduction. In some embodiments, the methods described herein comprise culturing the genetically modified cell (e.g., microbe) in a culture medium in a batch fermentation, such as the batch fermentation exemplified in Example 2 below. Various different culture mediums can be selected based on the particular transgenic species used and the growth conditions, among other things. In some embodiments, a defined culture medium is used. In some embodiments, minimal culture medium may be supplemented as needed to optimize growth and / or carotenoid production of a given genetically modified microbial cell type. For example, in some embodiments, such as those utilizing genetically modified .S', cerevisiae, the culture medium may comprise about l%-5% (e.g., 3%) w / v maltodextrin, about 0.05%-0.5% (e.g., 0.2%) w / v glucose, alphaamylase, or any combination thereof. In some embodiments, the culture medium may comprise yeast extract, e.g., 0.5-5 g / L (e.g., 2 g / L) of yeast extract, and / or an iron source. In some embodiments, the iron source is iron sulfate, e.g., iron sulfate heptahydrate. The iron sulfate may be at a concentration from 0.1 mM to 20 mM, from 0.5 mM to 10 mM, or from 1.0 mM to 5 mM, e.g., about 0.5 mM, 1.0 mM, about 1.2 mM, about 1.5 mM, about 1.7 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.4 mM, about 4 mM, about 4.5 mM, or about 5 mM.

[0199] The disclosed methods may further comprise extracting the produced carotenoid from the genetically modified cell (e.g., microbe). Various extraction methods known in the art can be used to extract carotenoid from the genetically modified cell (e.g., microbe), depending on the cell type. The extraction step may comprise permeabilizing the cell and / or solubilizing the produced carotenoids. Exemplary methods to extract carotenoids from yeast (e.g., .S'. cerevisiae) are shown in Example 1-10 below.

[0200] The disclosed methods of bioproduction may be further optimized and developed to increase yield. For example, in some embodiments, the disclosed methods may produce at least about 0. 1 pg / L, at least about 0.2 pg / L, at least about 0.3 pg / L, at least about 0.4 pg / L, at least about 0.5 pg / L, at least about 0.6 pg / L, at least about 0.7 pg / L, at least about 0.8 pg / L, at least about 0.9 pg / L, at least about 1.0 pg / L, at least about 1.1 pg / L, at least about 1.2 pg / L, at least about 1.3 pg / L, at least about 1.4 pg / L, at least about 1.5 pg / L, at least about 1.6 pg / L, at least about 1.7 pg / L, at least about 1.8 pg / L, at least about 1.9 pg / L, at least about 2.0 pg / L, at least about 2.1 pg / L, at least about 2.2 pg / L, at least about 2.3 pg / L, at least about 2.4 pg / L, at least about 2.5 pg / L, at least about 3.0 pg / L, at least about 3.5 pg / L, at least about 4.0 pg / L, at least about 4.5 pg / L, at least about 5.0 pg / L, at least about 5.5 pg / L, at least about 6.0 pg / L, at least about 6.5 pg / L, at least about 7.0 pg / L, at least about 7.5 pg / L, at least about 8.0 pg / L, at least about 8.5 pg / L, at least about 9.0 pg / L, at least about 9.5 pg / L, at least about 10.0 pg / L, at least about 20 pg / L, at least about 30 pg / L, at least about 40 pg / L, at least about 50 pg / L, at least about 75 pg / L, at least about 100 pg / L, at least about 150.0 pg / L, at least about 200.0 pg / L, at least about 250.0 pg / L, at least about 300.0 pg / L, at least about 350.0 pg / L, at least about 400.0 pg / L, at least about 450.0 pg / L, at least about 500.0 pg / L, at least about 600.0 pg / L, at least about 700.0 pg / L, at least about 800.0 pg / L, at least about 900.0 pg / L, at least about 1.00 mg / L, at least about 1.25 mg / L, at least about 1.50 mg / L, at least about 1.75 mg / L, at least about 2.00 mg / L, at least about 2.25 mg / L, at least about 2.50 mg / L, at least about 2.75 mg / L, at least about 3.00 mg / L, at least about 3.25 mg / L, at least about 3.50 mg / L, at least about 3.75 mg / L, at least about 4.00 mg / L, at least about 4.00 mg / L, at least about 4.25 mg / L, at least about 4.50 mg / L, at least about 4.75 mg / L, at least about 5.00 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about 10 mg / L, at least about 15 mg / L, at least about 20 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 35 mg / L, at least about 40 mg / L, at least about 45 mg / L, at least about 50 mg / L, at least about 55 mg / L, at least about 60 mg / L, at least about 65 mg / L, at least about 70 mg / L, at least about 75 mg / L, at least about 80 mg / L, at least about 85 mg / L, at least about 90 mg / L, at least about 95 mg / L, at least about 100 mg / L, at least about 105 mg / L, at least about 110 mg / L, at least about 115 mg / L, at least about 120 mg / L, at least about 125 mg / L, at least about 130 mg / L, at least about 135 mg / L, at least about 140 mg / L, at least about 145 mg / L, or at least about 150 mg / L or more of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof) within at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 36 hours, or at least about 48 hours of culture.

[0201] In some embodiments, the disclosed methods may produce at least about 0. 1 pg / L, at least about 0.2 pg / L, at least about 0.3 pg / L, at least about 0.4 pg / L, at least about 0.5 pg / L, at least about 0.6 pg / L, at least about 0.7 pg / L, at least about 0.8 pg / L, at least about 0.9 pg / L, at least about 1.0 pg / L, at least about 1.1 pg / L, at least about 1.2 pg / L, at least about 1.3 pg / L, at least about 1.4 pg / L, at least about 1.5 pg / L, at least about 1.6 pg / L, at least about 1.7 pg / L, at least about 1.8 pg / L, at least about 1.9 pg / L, at least about 2.0 pg / L, at least about 2. 1 pg / L, at least about 2.2 pg / L, at least about 2.3 pg / L, at least about 2.4 pg / L, at least about 2.5 pg / L, at least about 3.0 pg / L, at least about 3.5 pg / L, at least about 4.0 pg / L, at least about 4.5 pg / L, at least about 5.0 pg / L, at least about 5.5 pg / L, at least about 6.0 pg / L, at least about 6.5 pg / L, at least about 7.0 pg / L, at least about 7.5 pg / L, at least about 8.0 pg / L, at least about 8.5 pg / L, at least about 9.0 pg / L, at least about 9.5 pg / L, at least about 10.0 pg / L, at least about 20 pg / L, at least about 30 pg / L, at least about 40 pg / L, at least about 50 pg / L, at least about 75 pg / L, at least about 100 pg / L, at least about 150.0 pg / L, at least about 200.0 pg / L, at least about 250.0 pg / L, at least about 300.0 pg / L, at least about 350.0 pg / L, at least about 400.0 pg / L, at least about 450.0 pg / L, at least about 500.0 pg / L, at least about 600.0 pg / L, at least about 700.0 pg / L, at least about 800.0 pg / L, at least about 900.0 g / L, at least about 1.00 mg / L, at least about 1.25 mg / L, at least about 1.50 mg / L, at least about 1.75 mg / L, at least about 2.00 mg / L, at least about 2.25 mg / L, at least about 2.50 mg / L, at least about 2.75 mg / L, at least about 3.00 mg / L, at least about 3.25 mg / L, at least about 3.50 mg / L, at least about 3.75 mg / L, at least about 4.00 mg / L, at least about 4.00 mg / L, at least about 4.25 mg / L, at least about 4.50 mg / L, at least about 4.75 mg / L, at least about 5.00 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about 10 mg / L, at least about 15 mg / L, at least about 20 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 35 mg / L, at least about 40 mg / L, at least about 45 mg / L, at least about 50 mg / L, at least about 55 mg / L, at least about 60 mg / L, at least about 65 mg / L, at least about 70 mg / L, at least about 75 mg / L, at least about 80 mg / L, at least about 85 mg / L, at least about 90 mg / L, at least about 95 mg / L, at least about 100 mg / L, at least about 105 mg / L, at least about 110 mg / L, at least about 115 mg / L, at least about 120 mg / L, at least about 125 mg / L, at least about 130 mg / L, at least about 135 mg / L, at least about 140 mg / L, at least about 145 mg / L, or at least about 150 mg / L or more of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof) within about 6 hours of culture or less, about 12 hours of culture or less, about 18 hours of culture or less, about 24 hours of culture or less, about 36 hours of culture or less, or about 48 hours of culture or less.

[0202] In some embodiments, the present disclosure provides a method of increasing production of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof), comprising culturing the genetically modified cell (e.g., microbe) described herein in a culture medium. In some embodiments, the present disclosure provides a method of increasing production of zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and / or -cryptoxanthin, comprising culturing a genetically modified cell (e.g., microbe) disclosed herein in a culture medium. In some embodiments, the present disclosure provides a method of increasing production of zeaxanthin and / or astaxanthin, comprising culturing a genetically modified cell (e.g., microbe) disclosed herein in a culture medium.

[0203] In some embodiments, the disclosed methods may produce an increased level of a carotenoid (e.g., zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof) in a genetically modified cell (e.g., microbe) disclosed herein compared to methods of using a cell (e.g., microbe) having the same genetic background but without the transgene encoding the CBP or the genetic modification capable of increasing the expression of the CBP. In some embodiments, the disclosed methods may produce at least 1.1-fold, at least 1.2-fold. at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18- fold, at least 19-fold, or at least 20-fold of a carotenoid (e.g., zeaxanthin and / or astaxanthin) compared to methods of using a cell (e.g., microbe) having the same genetic background but without the transgene encoding the CBP or the genetic modification capable of increasing the expression of the CBP.

[0204] The present disclosure also provides methods of increasing a carotenoid production in a cell (e.g., microbe), comprising contacting the cell (e.g., microbe) with an agent that increases gene copy number, expression, and / or activity of a CBP.

[0205] The present disclosure also provides methods of increasing a carotenoid production in a cell (e.g., microbe), comprising contacting the cell (e.g., microbe) with an agent that increases gene copy number, expression, and / or activity of a CBP, wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof.

[0206] In some embodiments, provided herein is a method of increasing a carotenoid production in a cell (e.g., microbe), comprising contacting the cell (e.g., microbe) with an agent that increases gene copy number, expression, and / or activity of a CBP, wherein the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog, or human Aster-A (HsAsterA) or a human Aster-A homolog.

[0207] In some embodiments, provided herein is a method of increasing production of a carotenoid selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin in a cell (e.g., microbe), comprising contacting the cell (e.g., microbe) with an agent that increases gene copy number, expression, and / or activity of a CBP.

[0208] In some embodiments, provided herein is a method of increasing production of zeaxanthin and / or astaxanthin in a cell (e.g., microbe), comprising contacting the cell (e.g., microbe) with an agent that increases gene copy number, expression, and / or activity of a CBP.

[0209] In some embodiments, the agent used in the methods disclosed herein is a nucleic acid (e.g., an expression vector encoding a CBP), a protein, and / or a small molecule.

[0210] The present disclosure also provides batches of carotenoid produced by the methods disclosed herein. A bioproduction batch of carotenoid may have a chemical purity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or any values in between any of the two aforementioned values, and no single impurity of greater than 1%, no greater than about 0.5%, or greater than about 0.1%. The level of impurities in a given batch of carotenoid can be determined by high-performance liquid chromatography (HPLC) and other suitable techniques.

[0211] Methods of Making Genetically Modified Microbes

[0212] In some embodiments, the present disclosure provides a method of making a genetically modified cell (e.g., microbe) described herein, comprising contacting the cell (e.g., microbe) with a nucleic acid (e.g., a DNA or an RNA) encoding a ferredoxin protein. The ferredoxin protein may be any ferredoxin protein disclosed herein (e.g., the ferredoxin proteins described in the sections above). In some embodiment, the nucleic acid is a vector (e.g., an expression vector). Technologies to introduce / deliver nucleic acids to various microbes are well known in the art and can be used in the methods provided herein.

[0213] In some embodiments, the present disclosure provides a method of making a genetically modified cell (e.g., microbe) described herein, comprising contacting the cell (e.g., microbe) with a nucleic acid (e.g., a DNA or an RNA) encoding a CBP. The CBP may be any CBP disclosed herein (e.g., the CBPs described in the sections above). In some embodiments, the CBP is a heterologous CBP. In some embodiments, the nucleic acid is a vector (e.g., an expression vector). Technologies to introduce / deliver nucleic acids to various microbes are well known in the art and can be used in the methods provided herein.

[0214] Examples of Implementations of the Present Disclosure

[0215] Specific implementations of the present disclosure include, but are not limited to, the following:

[0216] Implementation 1. A genetically modified cell comprising (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a transgene that encodes a native ferredoxin protein, wherein the transgene enhances the production of the carotenoid.

[0217] Implementation 2. The genetically modified cell of implementation 1, wherein the genetically modified cell is a genetically modified microbe.

[0218] Implementation 3. The genetically modified cell of implementation 2, wherein the genetically modified microbe is a bacterium. Implementation 4. The genetically modified cell of implementation 3, wherein the bacterium is Escherichia coli (E. coli).

[0219] Implementation 5. The genetically modified cell of implementation 2, wherein the genetically modified microbe is a yeast.

[0220] Implementation 6. The genetically modified cell of implementation 5, wherein the yeast is Saccharomyces cerevisiae (S. cerevisiae), Yarrowia lipolytica, Pichia pastoris, or Kluyveromyces marxianus.

[0221] Implementation 7. The genetically modified cell of implementation 5, wherein the yeast is not Saccharomyces cerevisiae (S. cerevisiae).

[0222] Implementation 8. The genetically modified cell of any one of implementations 1-7, wherein the transgene is integrated into the genome of the genetically modified cell.

[0223] Implementation 9. The genetically modified cell of any one of implementations 1-2 and 5- 8, wherein the genetically modified cell is a yeast, and the transgene is integrated into the ARS1021 locus.

[0224] Implementation 10. The genetically modified cell of any one of implementations 1-7, wherein the transgene is not integrated into the genome of the genetically modified cell.

[0225] Implementation 11. The genetically modified cell of any one of implementations 1-10, wherein the ferredoxin protein is a wild-type ferredoxin protein.

[0226] Implementation 12. The genetically modified cell of any one of implementations 1-10, wherein the ferredoxin protein is an engineered ferredoxin protein.

[0227] Implementation 13. The genetically modified cell of any one of implementations 1-12, wherein the ferredoxin protein is selected from APD1, ATM1, YAH1, ILV3, NAR1, DRE2, and TAH18.

[0228] Implementation 14. The genetically modified cell of any one of implementations 1-13, wherein the ferredoxin protein comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, and 10.

[0229] Implementation 15. The genetically modified cell of any one of implementations 1-14, wherein the transgene comprises a nucleotide sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to a nucleotide sequence selected from SEQ ID NOs: 1, 3, 5, 7, and 9.

[0230] Implementation 16. The genetically modified cell of any one of implementations 1-12, wherein the ferredoxin protein is not YAH1 or ferredoxin-3.

[0231] Implementation 17. The genetically modified cell of any one of implementations 1-12 and 16, wherein the ferredoxin protein is selected from APD1, ATM1, ILV3, and NAR1.

[0232] Implementation 18. The genetically modified cell of any one of implementations 1-12 and 16-17, wherein the ferredoxin protein comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 2, 4, 8, and 10.

[0233] Implementation 19. The genetically modified cell of any one of implementations 1-12 and 16-18, wherein the transgene comprises a nucleotide sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to a nucleotide sequence selected from SEQ ID NOs: 1, 3, 7, and 9.

[0234] Implementation 20. The genetically modified cell of any one of implementations 1-2 and 5- 19, wherein the genetically modified cell is a yeast, and the transgene further comprises pCCW12 promoter and / or tYOL036W terminator.

[0235] Implementation 21. The genetically modified cell of any one of implementations 1-20, wherein the genetically modified cell comprises two or more transgenes that encode ferredoxin proteins.

[0236] Implementation 22. The genetically modified cell of implementation 21, wherein the two or more transgenes encode the same ferredoxin protein.

[0237] Implementation 23. The genetically modified cell of implementation 21, wherein the two or more transgenes encode different ferredoxin proteins.

[0238] Implementation 24. The genetically modified cell of implementation 23, wherein the two or more transgenes encode different ferredoxin proteins selected from APD1, ATM1, YAH1, ILV3, and NAR1.

[0239] Implementation 25. The genetically modified cell of implementation 24, wherein the two or more transgenes encode ferredoxin proteins comprising APD1 and ATM1, or ATM1 and YAH1. Implementation 26. The genetically modified cell of any one of implementations 1-25, wherein the heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid is selected from CrtE, CarRP, CarB, and / or CrtZ.

[0240] Implementation 27. The genetically modified cell of implementation 26, wherein the CrtZ gene is not Saccharolobits solfataricus CrtZ (SsCrtZ).

[0241] Implementation 28. The genetically modified cell of any one of implementations 1-27, wherein the cell is capable of producing an increased level of carotenoid when cultured in a culture medium comprising an iron source.

[0242] Implementation 29. The genetically modified cell of implementation 28, wherein the iron source is iron sulfate.

[0243] Implementation 30. The genetically modified cell of any one of implementations 1-29, wherein the production of the carotenoid is enhanced by at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, or at least 1.6-fold.

[0244] Implementation 31. The genetically modified cell of any one of implementations 1-30, wherein the carotenoid is zeaxanthin or a derivative thereof.

[0245] Implementation 32. The genetically modified cell of implementation 31 , wherein the derivative of zeaxanthin is not violaxanthin.

[0246] Implementation 33. The genetically modified cell of implementation 31 or 32, wherein the carotenoid is selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin.

[0247] Implementation 34. The genetically modified cell of any one of implementations 31-33, wherein the carotenoid is zeaxanthin or astaxanthin.

[0248] Implementation 35. A genetically modified cell comprising a transgene that encodes a native ferredoxin protein, wherein the genetically modified cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the native ferredoxin protein, optionally wherein the genetically modified cell is a genetically modified microbe.

[0249] Implementation 36. A genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the ferredoxin protein is not YAH1 or ferredoxin-3, optionally wherein the genetically modified cell is a genetically modified microbe. Implementation 37. The genetically modified cell of implementation 36, wherein the ferredoxin protein is selected from APD1, ATM1, ILV3, and NAR1.

[0250] Implementation 38. A genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the ferredoxin protein, wherein the carotenoid is not violaxanthin, optionally wherein the genetically modified cell is a genetically modified microbe.

[0251] Implementation 39. The genetically modified cell of implementation 38, wherein the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or betacryptoxanthin.

[0252] Implementation 40. The genetically modified cell of implementation 38 or 39, wherein the carotenoid is zeaxanthin or astaxanthin.

[0253] Implementation 41. A method of producing a carotenoid, comprising culturing the genetically modified cell of any one of implementations 1-40 in a culture medium.

[0254] Implementation 42. The method of implementation 41, wherein the method comprises culturing the genetically modified cell of any one of claims 1-40 in a culture medium in a batch fermentation.

[0255] Implementation 43. The method of implementation 41 or 42, wherein the culture medium comprises an iron source.

[0256] Implementation 44. The method of implementation 43, wherein the iron source is iron sulfate.

[0257] Implementation 45. The method of any one of implementations 41-44, wherein the method further comprises extracting the carotenoid from the genetically modified cell.

[0258] Implementation 46. The method of any one of implementations 41-45, wherein the carotenoid is zeaxanthin or a derivative thereof.

[0259] Implementation 47. The method of implementation 46, wherein the derivative of zeaxanthin is not violaxanthin.

[0260] Implementation 48. The method of implementation 46 or 47, wherein the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin. Implementation 49. The method of any one of implementations 46-48, wherein the carotenoid is zeaxanthin or astaxanthin.

[0261] Implementation 50. A method of increasing production of zeaxanthin or a derivative thereof, wherein the derivative of zeaxanthin is not violaxanthin, comprising culturing the genetically modified cell of any one of implementations 1 -40 in a culture medium.

[0262] Implementation 51. A method of making a genetically modified cell of any one of implementations 1-40, comprising contacting the cell with a nucleic acid encoding a ferredoxin protein.

[0263] Implementation 52. The method of implementation 51, wherein the nucleic acid is an expression vector.

[0264] Implementation 53. A method of increasing a carotenoid production in a cell, comprising contacting the cell with an agent that increases gene copy number, expression, and / or activity of ferredoxin, wherein the ferredoxin is not YAH1 or ferredoxin-3, optionally wherein the genetically modified cell is a genetically modified microbe.

[0265] Implementation 54. The method of implementation 53, wherein the ferredoxin is selected from APD1, ATM1, ILV3, and NAR1.

[0266] Implementation 55. A method of increasing production of a carotenoid in a cell, wherein the carotenoid is not violaxanthin, comprising contacting the cell with an agent that increases gene copy number, expression, and / or activity of ferredoxin, optionally wherein the genetically modified cell is a genetically modified microbe.

[0267] Implementation 56. The method of implementation 55, wherein the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin.

[0268] Implementation 57. The method of implementation 55 or 56, wherein the carotenoid is zeaxanthin or astaxanthin.

[0269] Implementation 58. The method of any one of implementations 53-57, wherein the agent is a nucleic acid, a protein, and / or a small molecule.

[0270] Implementation 59. The method of any one of implementations 53-58, wherein the agent is a nucleic acid encoding a ferredoxin protein.

[0271] Implementation 60. A genetically modified cell comprising (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a transgene that encodes a heterologous carotenoid binding protein (CBP), wherein the transgene enhances the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof.

[0272] Implementation 61. A genetically modified cell comprising (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a genetic modification capable of increasing the expression of a carotenoid binding protein (CBP), wherein the genetic modification enhances the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof.

[0273] Implementation 62. The genetically modified cell of implementation 60 or 61, wherein the genetically modified cell is a genetically modified microbe.

[0274] Implementation 63. The genetically modified cell of implementation 62, wherein the genetically modified microbe is a bacterium.

[0275] Implementation 64. The genetically modified cell of implementation 63, wherein the bacterium is Escherichia coli (E. colt).

[0276] Implementation 65. The genetically modified cell of implementation 62, wherein the genetically modified microbe is a yeast.

[0277] Implementation 66. The genetically modified cell of implementation 65, wherein the yeast is Saccharomyces cerevisiae (S. cerevisiae), Yarrowia lipolytica, Pichia pastoris, or Kluyveromyces marxianus.

[0278] Implementation 67. The genetically modified cell of implementation 65, wherein the yeast is not Saccharomyces cerevisiae (S. cerevisiae).

[0279] Implementation 68. The genetically modified cell of any one of implementations 60 and 62-67, wherein the transgene is integrated into the genome of the genetically modified cell.

[0280] Implementation 69. The genetically modified cell of any one of implementations 60, 62 and 65-68, wherein the genetically modified cell is a yeast, and the transgene is integrated into the ARS511 locus.

[0281] Implementation 70. The genetically modified cell of any one of implementations 60 and 62-67, wherein the transgene is not integrated into the genome of the genetically modified cell. Implementation 71. The genetically modified cell of any one of implementations 60-70, wherein the CBP is a wild-type CBP.

[0282] 72. The genetically modified cell of any one of implementations 60-70, wherein the CBP is an engineered CBP.

[0283] Implementation 73. The genetically modified cell of any one of implementations 60-72, wherein the CBP is selected from the steroidogenic acute regulatory lipid transfer (START) protein family or the Aster family of proteins.

[0284] Implementation 74. The genetically modified cell of any one of implementations 60-73, wherein the CBP is selected from the START protein family.

[0285] Implementation 75. The genetically modified cell of implementation 74, wherein the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog.

[0286] Implementation 76. The genetically modified cell of implementation 75, wherein the CBP is Bombyx mori CBP.

[0287] Implementation 77. The genetically modified cell of any one of implementations 60-76, wherein the CBP comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 12.

[0288] Implementation 78. The genetically modified cell of any one of implementations 60 and 62-77, wherein the transgene comprises a nucleotide sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 11.

[0289] Implementation 79. The genetically modified cell of any one of implementations 60-73, wherein the CBP is selected from the Aster family of proteins.

[0290] Implementation 80. The genetically modified cell of implementation 79, wherein the CBP is selected from Aster-A, Aster -B, and Aster -C, or homologs thereof.

[0291] Implementation 81. The genetically modified cell of implementation 80, wherein the CBP is human Aster-A, or a human Aster-A homolog.

[0292] Implementation 82. The genetically modified cell of any one of implementations 60-73 and 79-81, wherein the CBP comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 14. Implementation 83. The genetically modified cell of any one of implementations 60, 62-72, and 79-82, wherein the transgene comprises a nucleotide sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 13.

[0293] Implementation 84. The genetically modified cell of any one of implementations 60-72, wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof.

[0294] Implementation 85. The genetically modified cell of any one of implementations 60, 62 and 65-84, wherein the genetically modified cell is a yeast, and the transgene further comprises pGAL7 promoter and / or tHSP26 terminator.

[0295] Implementation 86. The genetically modified cell of any one of implementations 60-85, wherein the heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid is selected from CrtE, CarRP, CarB, and / or CrtZ.

[0296] Implementation 87. The genetically modified cell of any one of implementations 60-86, wherein the production of zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is enhanced by at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2- fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold.

[0297] Implementation 88. The genetically modified cell of any one of implementations 60-87, wherein the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and betacryptoxanthin.

[0298] Implementation 89. The genetically modified cell of any one of implementations 60-88, wherein the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is zeaxanthin or astaxanthin.

[0299] Implementation 90. A genetically modified cell comprising a transgene that encodes a heterologous carotenoid binding protein (CBP), wherein the genetically modified cell is capable of producing an increased level of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof, compared to a cell having the same genetic background but without the transgene encoding the heterologous carotenoid binding protein (CBP), optionally wherein the genetically modified cell is a genetically modified microbe.

[0300] Implementation 91. A genetically modified cell comprising a transgene that encodes a heterologous carotenoid binding protein (CBP), wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof, optionally wherein the genetically modified cell is a genetically modified microbe.

[0301] Implementation 92. The genetically modified cell of implementation 91, wherein the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog, or human Aster-A or a human Aster-A homolog.

[0302] Implementation 93. A method of producing (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof, comprising culturing the genetically modified cell of any one of claims 1-33 in a culture medium.

[0303] Implementation 94. The method of implementation 93, wherein the method comprises culturing the genetically modified cell of any one of claims 1-33 in a culture medium in a batch fermentation.

[0304] Implementation 95. The method of implementation 93 or 94, wherein the culture medium comprises an iron source.

[0305] Implementation 96. The method of implementation 95, wherein the iron source is iron sulfate.

[0306] Implementation 97. The method of any one of implementations 93-96, wherein the method further comprises extracting the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof from the genetically modified cell.

[0307] Implementation 98. The method of implementation 97, wherein the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin.

[0308] Implementation 99. The method of implementation 97 or 98, wherein the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is zeaxanthin or astaxanthin.

[0309] Implementation 100. A method of making a genetically modified cell of any one of implementations 61 and 62-92, comprising contacting the cell with a nucleic acid encoding a heterologous carotenoid binding protein (CBP). Implementation 101. The method of implementation 100, wherein the nucleic acid is an expression vector.

[0310] Implementation 102. A method of increasing the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof in a cell, comprising contacting the cell with an agent that increases gene copy number, expression, and / or activity of a heterologous carotenoid binding protein (CBP), wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof, optionally wherein the genetically modified cell is a genetically modified microbe.

[0311] Implementation 103. The method of implementation 102, wherein the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog, or human Aster-A or a human Aster- A homolog.

[0312] Implementation 104. The method of implementation 102 or 103, wherein the agent is a nucleic acid, a protein, and / or a small molecule.

[0313] Implementation 105. The method of any one of implementations 102-104, wherein the agent is a nucleic acid encoding a heterologous CBP.

[0314] EXAMPLES

[0315] The disclosure now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the scope of the disclosure in any way.

[0316] Example 1: Generation and Identification of Modified Yeast Strains with Ferredoxin Overexpression and Increased Carotenoid Production

[0317] This Example describes generation of genetically modified 5. cerevisiae strains overexpressing native ferredoxin genes (APD1, ATM1, YAH1, ILV3, and NAR1), and evaluation of their production of carotenoids in a 96- well plate screening.

[0318] Individual ferredoxin genes (APD1, ATM1, YAH1, ILV3, and NAR1 of SEQ ID NOS: 1, 3, 5, 7, and 9, respectively) were expressed from the ARS1021 locus driven by pCCW12 promoter and IYOL036W terminator from a .S', cerevisiae parent strain (STR2820, STR3142, or STR3145). Genomic integrations were performed using a lithium acetate-PEG- salmon sperm DNA chemical transformation method with a Cas 12-based system for directed nuclease-guided editing. These S. cerevisiae parent strains were previously engineered to express variants of CrtE, CarRP, CarB and CrtZ, which are genes in the CRT gene cluster responsible for the biosynthesis of carotenoids and encode geranylgeranyl diphosphate synthase, bifunctional lycopene cyclase / phytoene synthase, phytoene desaturase, and P- carotene hydroxylase, respectively. The strains also contain one or more additional genetic modifications. Edits and genetic information of parent .S'. cerevisiae strains are described in Table 4.

[0319] Following ferredoxin gene overexpression, S. cerevisiae strains were grown and assayed at 30°C in 96 mid- well plates in YPD (yeast extract peptone dextrose) media for 24- 48 hours, before transfer into defined media containing 3% maltodextrin, 0.2% glucose defined media (modified from Westfall et al. (2012) Proc Natl Acad Sci U S A. 1O9(3):E111-E118) with alpha-amylase for 44 hours.

[0320] For plate-based extraction, an aliquot of whole cell broth was dispensed into dimethyl sulfoxide and agitated to effect cell permeabilization and solubilization of the carotenoids. A secondary dilution was then performed into a methanol and acetone solution as a sample matrix for analysis on an Agilent 1290 Infinity II UHPLC. Separation of a 5 pL sample injection was performed via a 2.1 x 50 mm C18 Agilent ZORBAX RRHD Eclipse Plus analytical column and a stationary phase matched 2.1 x 5 mm guard column. The mobile phase was maintained isocratic at 80:15:5% composition of methanol:acetone:tetrahydrofuran containing 0.1 % butylated hydroxytoluene but with a flow gradient ranging from 0.4 to 1.1 mL / min. Detection of zeaxanthin and beta-carotene was performed at 476 nm and confirmed per retention time via authentic chemical standards.

[0321] As shown in FIGs. 1A and 1C, overexpressing individual ferredoxin genes APD1, ATM1, YAH1, ILV3, or NAR1 in parent strains STR2820 and STR3142 resulted in increased zeaxanthin production. With regard to the intermediate product beta-carotene, overexpressing individual ferredoxin genes APD1 , ATM1 , YAH1 , ILV3, or NAR1 resulted in decreased production of beta-carotene (FIGs. IB and ID). Taken together, these data show that overexpressing ferredoxin genes increased the production of zeaxanthin in .S', cerevisiae. With respect to parent strain STR3145 and its child strains, overexpressing individual ferredoxin genes APD1, ATM1, YAH1, ILV3, or NAR1 resulted in variable levels of productions of zeaxanthin (FIG. IE) and beta-carotene (FIG. IF). Without wishing to be bound by any particular scientific theory, strain STR3145 and its child strains (STR3431, STR3433, STR3434, STR3435, and STR3436) express a distinct CrtZ variant (Saccharolobus solfataricus CrtZ), which may affect the production of zeaxanthin and beta- carotene independently or in conjunction with the overexpressed ferredoxin genes (e.g., SsCrtZ may lead to the plateauing effect of carotenoid production).

[0322] Example 2: Co-overexpression of Ferredoxin Proteins in Modified Yeast Strains Increased Carotenoid Production

[0323] This Example describes co-overexpressing native ferredoxin genes in S. cerevisiae strains and the evaluation of the production of carotenoids in the strains.

[0324] Individual or combinations of ferredoxin genes were expressed in a .S'. cerevisiae parent strain (STR3334) as in above Example 1. Following the expression of individual or combinations of ferredoxin genes, .S', cerevisiae were grown and assayed in 96 mid-well plates as in above Example 1. Edits and genetic information of parent .S. cerevisiae strains are described in Table 4.

[0325] As shown in FIG. 2A, overexpression of APD1, overexpression of YAH1, co- overexpression of APD1 and ATM1, co-overexpression of ATM1 and YAH1, and co- overexpression of APD1, ATM1, and YAH1 resulted in increased zeaxanthin production in child strains compared to the zeaxanthin production level in the S. cerevisiae parent strain STR3334 without overexpression. With regard to the intermediate product beta-carotene, co- overexpression of ATM1 and YAH1 resulted in increased production of beta-carotene, while overexpression of APD1, overexpression of YAH1, co-overexpression of APD1 and ATM1, or co-overexpression of APD1, ATM1, and YAH1 resulted in decreased production of betacarotene (FIG. 2B). Taken together, these data show that co-overexpressing ferredoxin genes increased the production of zeaxanthin in S. cerevisiae.

[0326] Example 3: Overexpressing Ferredoxin Genes in Yeast Resulted in Increased Production of Zeaxanthin in Batch Fermentation

[0327] This example describes overexpressing native ferredoxin genes (APD1, ATM1, YAH1, ILV3, and NAR1) in S. cerevisiae strains as in above Example 1 to increase the production of zeaxanthin in a batch fermentation process. Edits and genetic information of parent .S'. cerevisiae strains are described in Table 4.

[0328] Briefly, 1 mL of glycerol stock of S. cerevisiae strain stored at -80 °C freezer was inoculated into 50 mL of either minimal media (control seed media) or fortified minimal media (fortified seed media) in a 250 mL baffled flask. After 24 hours of incubation with an OD greater than 2, 0.5 mL of the culture from the first seed was passaged into a second seed flask containing 50 mL control media or fortified media in a 250 mL baffled flask. Second shake flask was incubated for 24 hours with the final expected target OD greater than 6. Cultivation conditions for both seed flasks were as follows: temperature was controlled at 30 °C, agitation at 225 rpm in a 1 inch throw shaker to allow enough oxygenation. Minimal media (control seed media) was previously published in Westfall et al. (2012) Proc Natl Acad Sci U S A. 1O9(3):E111-E118. Fortified seed media had the exact composition as published in Westfall et al. except the addition of 2 g / L of yeast extract and 1.7 mM of iron sulfate heptahydrate.

[0329] Multiple 250 mL bioreactors were used for this Example. Each bioreactor prior to inoculation was batched with either 90 mL of minimal batch media or fortified batch media. Culture from the end of seed 2 was used as an inoculum. Each bioreactor was then inoculated with 10 mL of diluted culture to reach an initial OD of 0.6. The temperature in the reactor was controlled at 30 °C and pH at 5.0 using 14% ammonium hydroxide. The dissolved oxygen was controlled at 30% by cascading agitation (from 600-2500 rpm) and then airflow (0.5-1 vvm). The initial glucose concentration in the batch phase is 19.5 g / L. The end of batch phase was evident by the second DO spike which coincided with the exhaustion of batch glucose along with any produced ethanol. After the batch phase, an exponential feed phase started. The starting feed rate was 0.01185 mL / min, with a volume basis of 100 mL initial volume. The growth rate of the feed was 0.15 / hr, and the exponential feed continued for 4.5 or until OUR was >150 mmol / L / h. Once either condition was met, exponential feed phase was ended and a constant low flow feed of 1 g_glucose / L_current_volume / hr was turned on. At the same time, DO spike bolus feeding was turned on. The boluses fed 6 g_glucose / L_current_volume over 60 minutes at a rate of 6 g_glucose / L_current_volume / hr. Both feeds were based on current volumes in the bioreactors. The control feed media contained 585 g / L dextrose, 2.25 g / L potassium phosphate monobasic, and 18 mL / L vitamin stock solution. Fortified feed media addition of 17 mM iron sulfate heptahydrate. Cell pellet and whole cell broth samples were taken from each reactor every 24 hours and stored at -80 °C freezer until analysis time. These samples were later used for product extraction and quantification.

[0330] Main batch fermentation media was modified from a recipe previously published in Westfall et al.. The minimal batch media for the fermentation was based on the Westfall recipe with modified concentrations. The fortified batch media had addition of 3.4 mM of iron sulfate heptahydrate. For zeaxanthin and beta-carotene detection, bioreactor derived fermentation whole cell broth was centrifuged with supernatant removed. A dimethyl sulfoxide and tetrahydrofuran solution was added to the resultant cell pellet and agitated to effect cell permeabilization and solubilization of the carotenoids. A secondary dilution was then performed into a methanol, acetone and tetrahydrofuran solution as a sample matrix for analysis on an Agilent 1290 Infinity II UHPLC. Separation of a 5 pL sample injection was performed via a 2.1 x 50 mm Cl 8 Agilent ZORBAX RRHD Eclipse Plus analytical column and stationary phase matched 2.1 x 5 mm guard column. The mobile phase was maintained isocratic at 80:15:5% composition of methanol: acetone: tetrahydrofuran containing 0.1 % butylated hydroxytoluene but with a flow gradient ranging from 0.2 to 0.6 mL / min. Detection of both analytes was performed at 476 nm and confirmed per retention time via authentic chemical standards.

[0331] As shown in FIG. 3A, overexpressing APD, ILV3, NAR1, or YAH1 resulted in consistently increased production of zeaxanthin over time when compared to .S', cerevisiae parent strains without overexpression. Overexpressing APD, ILV3, NAR1, or YAH1 did not result in significant changes in the production of beta-carotene (FIG. 3B). These data show that overexpressing ferredoxin genes increased the production of zeaxanthin in a batch fermentation process.

[0332] Example 4: Iron Supplementation in Media Resulted in Increased Production of Zeaxanthin in Yeast by Batch Fermentation

[0333] This example describes supplementing the culture media (i.e., seed media, batch media, and / or feed media) with iron to increase the production of zeaxanthin in S. cerevisiae in a batch fermentation process as described in above Example 3. Edits and genetic information of parent .S', cerevisiae strains are described in Table 4.

[0334] S. cerevisiae parent strains without overexpression of ferredoxin proteins (STR3072, STR3204, and STR3334) and child strains overexpressing native ferredoxin genes (STR3383 overexpressing APD1 and STR3386 overexpressing YAH1) were cultured according to the batch fermentation process as described in above Example 3. To evaluate the effect of iron supplementation on the production of zeaxanthin, iron sulfate heptahydrate was added to the seed media, batch media, and / or feed media used in the batch fermentation process.

[0335] FIG. 4 shows production of zeaxanthin in the above S. cerevisiae strains cultured with different media with or without iron addition as listed in Table 3. Table 3. Media Condition for Various Processes shown in FIG. 4.

[0336] As shown in FIG. 4, supplementing culture media with iron resulted in increased production of zeaxanthin over time in both control .S', cerevisiae strains without overexpression of ferredoxin genes (STR3072, STR3204, and STR 3334) and in ferredoxin- overexpressing strains (STR3383 and STR3386) when compared to conditions without iron supplementation.

[0337] These data show that iron supplementation in culture media increased the production of zeaxanthin in yeast in a batch fermentation process.

[0338] Example 5: Further Experimentation on Overexpression or Co-overexpression of Ferredoxin Proteins in Modified Yeast Strains in Plate Screening

[0339] This Example describes overexpressing or co-overexpressing native ferredoxin genes in additional S. cerevisiae strains and the evaluation of the production of carotenoids in the strains.

[0340] Individual or combinations of ferredoxin genes (APD1, ATM1, YAH1, ILV3, NAR1, DRE2, and TAH18) were expressed in S. cerevisiae parent strains (STR3072, STR3496, STR3519, STR3780, STR4002, STR4114, STR4344) as in above Example 1. Following the expression of individual or combinations of ferredoxin genes, .S'. cerevisiae parent and child strains were grown and assayed in 96 mid- well plates as in above Example 1. Edits and genetic information of parent S. cerevisiae strains are described in Table 4.

[0341] As shown in FIG. 5A, overexpression of individual ferredoxin genes APD1, ATM1, ILV3, NAR1, or YAH1 in child strains of parent STR3072 resulted in increased zeaxanthin production. With regard to the intermediate product beta-carotene, overexpression of individual ferredoxin genes resulted in comparable or reduced production of beta-carotene in the child strains compared to the level in the 5. cerevisiae parent strain STR3072 without overexpression (FIG. 5B).

[0342] Overexpression of individual ferredoxin genes ATM1 or YAH1, or the combination of APD1, ATM1, and YAH1 in child strains of parent STR3496 resulted in decreased zeaxanthin production (FIG. 5C) and decreased production of the intermediate product betacarotene (FIG. 5D) Without wishing to be bound by any particular scientific theory, strain STR3496 and its child strains (STR3638, STR3642, and STR3643) express a distinct CrtZ variant (Saccharolobus solfataricus CrtZ), which may affect the production of zeaxanthin and beta-carotene independently or in conjunction with the overexpressed ferredoxin genes (e.g., SsCrtZ may lead to the plateauing effect of carotenoid production).

[0343] As shown in FIG. 5E, overexpression of individual ferredoxin genes APD1 or NAR1, or the combination of DRE2 and TAH18, resulted in increased zeaxanthin production in child strains of parent STR3519. With regard to the intermediate product beta-carotene, overexpression of APD1 or NAR1, or the combination of DRE2 and TAH18, resulted in reduced production of beta-carotene in the child strains (FIG. 5F).

[0344] Overexpression of ferredoxin gene APD1 in child strain STR3994 of parent strain STR3780 resulted in increased zeaxanthin production (FIG. 5G) and increased production of the intermediate product beta-carotene (FIG. 5H).

[0345] As shown in FIG. 51, overexpression of ferredoxin gene NAR1 in parent strain STR4002 resulted in decreased or comparable zeaxanthin production, while overexpression of APD1 resulted in increased zeaxanthin production. With regard to the intermediate product beta-carotene, overexpression of APD1 or NAR1 resulted in reduced production of betacarotene in the child strains (FIG. 5 J). Without wishing to be bound by any particular scientific theory, strain STR4002 and its child strains (STR4121 and STR4122) express a distinct CrtZ variant (Saccharolobns solfataricus CrtZ), which may affect the production of zeaxanthin and beta-carotene independently or in conjunction with the overexpressed ferredoxin genes (e.g., SsCrtZ may lead to the plateauing effect of carotenoid production).

[0346] Overexpression of ferredoxin gene APD 1 in child strain STR4247 of parent strain STR4114 resulted in comparable levels of production of both zeaxanthin (FIG. 5K) and the intermediate product beta-carotene (FIG. 5L). Without wishing to be bound by any particular scientific theory, parent strain STR4114 was edited to express ferredoxin genes DRE2 and TAH18, where additionally expressing APD1 might not further affect the production of zeaxanthin and beta-carotene.

[0347] Overexpression of ferredoxin gene APD 1 in parent strain STR4344, which does not express the crtZ gene for the biosynthesis of carotenoids, did not affect the production of beta-carotene (FIG. 6).

[0348] These data provide additional verification of expression of ferredoxin genes and the effects on the production of zeaxanthin and beta-carotene.

[0349] Example 6: Overexpression of Ferredoxin Protein in Modified Yeast Strains Decreased Astaxanthin Production

[0350] This Example describes overexpressing native ferredoxin genes in 5. cerevisiae strains and the evaluation of the production of astaxanthin in the strains.

[0351] APD1 was expressed in S. cerevisiae parent strains (STR4873 and STR5249) as in above Example 1. Strains STR4873 and STR5249 were additionally modified to express CrtW gene for synthesis of astaxanthin. Following the expression of APD1, S. cerevisiae strains were grown and assayed in 96 mid- well plates as in above Example 1. Edits and genetic information of parent S. cerevisiae strains are described in Table 4.

[0352] Overexpression of APD1 resulted in decreased astaxanthin production in child strains (FIGs. 7A and 7C), comparable or increased production of beta-carotene in child strain STR5443 (FIG. 7B), and decreased production of beta-carotene in child strain STR5458 (FIG. 7D).

[0353] These data show that overexpressing ferredoxin genes in strains specialized for astaxanthin resulted in decreased production of astaxanthin.

[0354] Example 7: Further Experimentation on Overexpression or Co-overexpression of Ferredoxin Proteins in Modified Yeast Strains in Batch Fermentation

[0355] This Example describes overexpressing native ferredoxin genes in additional S. cerevisiae strains and the evaluation of the production of carotenoids in the strains.

[0356] APD1 was expressed in S. cerevisiae parent strains (STR4002 and STR4114) as in above Example 3. Following the expression of ferredoxin genes, S. cerevisiae parent and child strains were grown and assayed in a batch fermentation process as described in above Example 3. Edits and genetic information of parent S. cerevisiae strains are described in Table 4. Overexpression of APD1 in child strains resulted in comparable levels of production of zeaxanthin (FIGs. 8A and 8C) and beta-carotene (FIGs. 8B and 8D) as compared to the respective productions in parent strains.

[0357] Example 8: Generation and Identification of Modified Yeast Strains with Carotenoid Binding Protein Overexpression and Increased Carotenoid Production

[0358] This Example describes the generation of genetically modified S. cerevisiae strains overexpressing a gene encoding a carotenoid binding protein (CBP) from Bombyx mori (BmCBP) represented by SEQ ID NO: 11, and evaluation of their production of carotenoids in a 96-well plate screening.

[0359] The BmCBP gene of SEQ ID NO: 11 was expressed from the ARS511 locus driven by pGAL7 promoter and tHSP26 terminator from a .S'. cerevisiae parent strain (STR3072). Genomic integrations were performed using a lithium acetate-PEG-salmon sperm DNA chemical transformation method with a Cas 12-based system for directed nuclease-guided editing. The 5. cerevisiae parent strain was previously engineered to express CrtE, CarRP, CarB and CrtZ, which are genes in the CRT gene cluster responsible for the biosynthesis of carotenoids and encode geranylgeranyl diphosphate synthase, bifunctional lycopene cyclase / phytoene synthase, phytoene desaturase, and (1-carotene hydroxylase, respectively. It also contains one or more additional genetic modifications. Edits and genetic information of parent .S'. cerevisiae strains are described in Table 4.

[0360] Following BmCBP gene overexpression, parent and child .S'. cerevisiae strains, and a negative control strain CEN.PK 113-7D, were grown and assayed at 30°C in 96 mid-well plates in YPD (yeast extract peptone dextrose) media for 24-48 hours, before transfer into defined media containing 3% maltodextrin, 0.2% glucose defined media (modified from Westfall et al. (2012) Proc Natl Acad Sci U S A. 1O9(3):E111-E118) with alpha-amylase for 44 hours.

[0361] For plate-based extraction, an aliquot of whole cell broth was dispensed into dimethyl sulfoxide and agitated to effect cell permeabilization and solubilization of the carotenoids. A secondary dilution was then performed into a methanol and acetone solution as a sample matrix for analysis on an Agilent 1290 Infinity II UHPLC. Separation of a 5 pL sample injection was performed via a 2. 1 x 50 mm C18 Agilent ZORBAX RRHD Eclipse Plus analytical column and a stationary phase matched 2. 1 x 5 mm guard column. The mobile phase was maintained isocratic at 80:15:5% composition of methanol: ace tone: tetrahydrofuran containing 0.1 % butylated hydroxytoluene but with a flow gradient ranging from 0.4 to 1. 1 mL / min. Detection of zeaxanthin was performed at 476 nm and confirmed per retention time via authentic chemical standards. Cell density was estimated by measuring the optical density at 600 nm (OD600).

[0362] As shown in FIG. 9, overexpressing the BmCBP gene resulted in increased zeaxanthin production by child strain STR3519 compared to zeaxanthin production levels in .S'. cerevisiae parent strain STR3072 without overexpression (levels indicated as dashed lines). With regard to OD600, overexpressing the BmCBP gene did not result in obvious changes in cell density or culture growth. Taken together, these data show that overexpressing the BmCBP gene increased the production of zeaxanthin in .S', cerevisiae.

[0363] Example 9: Overexpressing BmCBP Gene in Yeast Resulted in Increased Production of Zeaxanthin in Batch Fermentation

[0364] This Example describes the generation of genetically modified S. cerevisiae strains overexpressing a gene encoding a carotenoid binding protein (CBP) from Bombyx mori (BmCBP) represented by SEQ ID NO: 11, and evaluation of their production of carotenoids in a batch fermentation process.

[0365] The BmCBP gene of SEQ ID NO: 11 was expressed from the ARS511 locus driven by pGAL7 promoter and tHSP26 terminator from .S', cerevisiae parent strains (STR3072 and STR3575) to produce child strains STR3519 and STR3668, respectively. Genomic integrations were performed using a lithium acetate-PEG-salmon sperm DNA chemical transformation method with a Casl 2-based system for directed nuclease-guided editing. The S. cerevisiae parent strains were previously engineered to express CrtE, CarRP, CarB and CrtZ, which are genes in the CRT gene cluster responsible for the biosynthesis of carotenoids and encode geranylgeranyl diphosphate synthase, bifunctional lycopene cyclase / phytoene synthase, phytoene desaturase, and 0-carotene hydroxylase, respectively. They also contain one or more additional genetic modifications. Edits and genetic information of parent S. cerevisiae strains are described in Table 4.

[0366] Briefly, 1 mL of glycerol stock of .S'. cerevisiae strains stored at -80 °C freezer was inoculated into 50 mL of minimal media in a 250 mL baffled flask. After 24 hours of incubation with an OD greater than 2, 0.5 mL of the culture from the first seed was passaged into a second seed flask containing 50 mL minimal media in a 250 mL baffled flask. Second shake flask was incubated for 24 hours with the final expected target OD greater than 6. Cultivation conditions for both seed flasks were as follows: temperature was controlled at 30 °C, agitation at 225 rpm in a 1 inch throw shaker to allow enough oxygenation. Minimal media was previously published in Westfall et al. (2012) Proc Natl Acad Sci U S A. 1O9(3):E111-E118.

[0367] Multiple 250 mL bioreactors were used for this Example. Each bioreactor prior to inoculation was batched with 90 mL of minimal batch media. Culture from the end of seed 2 was used as an inoculum. Each bioreactor was then inoculated with 10 mL of diluted culture to reach an initial OD of 0.6. The temperature in the reactor was controlled at 30 °C and pH at 5.0 using 14% ammonium hydroxide. The dissolved oxygen was controlled at 30% by cascading agitation (from 600-2500 rpm) and then airflow (0.5-1 vvm). The initial glucose concentration in the batch phase is 19.5 g / L. The end of batch phase was evident by the second DO spike which coincided with the exhaustion of batch glucose along with any produced ethanol. After the batch phase, an exponential feed phase started. The starting feed rate was 0.01185 mL / min, with a volume basis of 100 mL initial volume. The growth rate of the feed was 0.15 / hr, and the exponential feed continued for 4.5 or until OUR was >150 mmol / L / h. Once either condition was met, exponential feed phase was ended and a constant low flow feed of 1 g_glucose / L_current_volume / hr was turned on. At the same time, DO spike bolus feeding was turned on. The boluses fed 6 g_glucose / L_current_volume over 60 minutes at a rate of 6 g_glucose / L_current_volume / hr. Both feeds were based on current volumes in the bioreactors. The minimal feed media contained 585 g / L dextrose, 2.25 g / L potassium phosphate monobasic, and 18 mL / L vitamin stock solution. Cell pellet and whole cell broth samples were taken from each reactor every 24 hours and stored at -80 °C freezer until analysis time. These samples were later used for product extraction and quantification.

[0368] Main batch fermentation media was modified from a recipe previously published in Westfall et al.. The minimal batch media for the fermentation was based on the Westfall recipe with modified concentrations.

[0369] The seed media, feed media, and batch fermentation media may additionally be fortified with an iron source. Fortified seed media has the exact composition as published in Westfall et al. except the addition of 2 g / L of yeast extract and 1.7 mM of iron sulfate heptahydrate. Fortified feed media contains an addition of 17 mM iron sulfate heptahydrate. Fortified batch media contains an addition of 3.4 mM iron sulfate heptahydrate.

[0370] For zeaxanthin and detection, bioreactor derived fermentation whole cell broth was centrifuged with supernatant removed. A dimethyl sulfoxide and tetrahydrofuran solution was added to the resultant cell pellet and agitated to effect cell permeabilization and solubilization of the carotenoids. A secondary dilution was then performed into a methanol, acetone and tetrahydrofuran solution as a sample matrix for analysis on an Agilent 1290 Infinity II UHPLC. Separation of a 5 L sample injection was performed via a 2.1 x 50 mm C18 Agilent ZORBAX RRHD Eclipse Plus analytical column and stationary phase matched 2.1 x 5 mm guard column. The mobile phase was maintained isocratic at 80: 15:5% composition of methanol: acetone: tetrahydrofuran containing 0.1 % butylated hydroxytoluene but with a flow gradient ranging from 0.2 to 0.6 mL / min. Detection of both analytes was performed at 476 nm and confirmed per retention time via authentic chemical standards.

[0371] As shown in FIG. 10, overexpressing BmCBP resulted in consistently increased production of zeaxanthin in child strains STR3519 and STR3668 over time when compared to the S. cerevisiae parent strains STR3072 and STR3575 without overexpression (~79% increase in titer relative to the parent strain in a 250 mL bioreactor). These data show that overexpressing the BmCBP gene increased the production of zeaxanthin in a batch fermentation process.

[0372] Example 10: Overexpressing HsAsterA Gene but not HgCRA2, HsApoB, or tHsApoB(29) in Yeast Resulted in Increased Production of Zeaxanthin in Batch Fermentation

[0373] This Example describes the generation of genetically modified 5. cerevisiae strains overexpressing a gene encoding HsAsterA, HgCRA2, human apolipoprotein B (HsApoB), or truncated human apolipoprotein B (tHsApoB(29)) having 29% of the length of the full-length HsApoB, and evaluation of their production of carotenoids in a batch fermentation process.

[0374] HsAsterA, HgCRA2, HsApoB, or tHsApoB(29) genes were expressed from the ARS511 locus from the .S', cerevisiae strain STR3992, a strain that overexpresses BmCBP, to replace BmCBP and to produce strains that express one of HsAsterA (STR4138), HgCRA2 (STR4232), HsApoB (STR4336), or tHsApoB(29)(STR4140). Zeaxanthin production in a batch fermentation process was evaluated for both strains as in above Example 9.

[0375] As shown in FIG. 11, overexpressing BmCBP or HsAsterA resulted in consistently higher production of zeaxanthin in respective 5. cerevisiae strains STR3922 and STR4138 over time when compared to the 5. cerevisiae strains STR4232 and STR4140, expressing HgCRA2 and tHSApoB, respectively (~150% increase in titer relative to STR4232 and STR4140 in a 250 mL bioreactor). Overexpressing BmCBP or HsAsterA resulted in similar levels of zeaxanthin production. These data show that overexpressing the BmCBP or HsAsterA gene, but not HgCRA2 or tHSApoB, increased the production of zeaxanthin in a batch fermentation process. As shown in FIG. 12, overexpressing HsApoB resulted in consistently decreased production of zeaxanthin in .S'. cerevisiae strain STR4336 over time when compared to the .S'. cerevisiae strain STR3922 which overexpresses BmCBP (>50% decrease in titer relative to STR3922). These data show that overexpressing HsApoB did not achieve enhanced zeaxanthin production as seen in BmCMP overexpression.

[0376] Table 4. Edits to Exemplified Parent S. cerevisiae Strains

[0377] * “Ps” stands for Paracoccus sp. “Pt” stands for Picrophilus torridus. “Ss” stands for Saccharolobus solfataricus . “Bt” stands for Blakeslea trispora. “Me” stands for Mucor circinelloides. “Xd” stands for Xanthophyllomyces dendrorhous.

[0378] Incorporation by Reference

[0379] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0380] Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.

[0381] Equivalents

[0382] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A genetically modified cell comprising (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) at least one transgene that encodes a native ferredoxin protein, wherein the transgene enhances the production of the carotenoid.

2. The genetically modified cell of claim 1 , wherein the genetically modified cell is a genetically modified microbe, optionally wherein the genetically modified microbe is a bacterium or a yeast.

3. The genetically modified cell of claim 1 or 2, wherein the ferredoxin protein is a wildtype ferredoxin protein or an engineered ferredoxin protein.

4. The genetically modified cell of any one of claims 1-3, wherein the ferredoxin protein is selected from APD1, ATM1, YAH1, ILV3, and NAR1.

5. The genetically modified cell of any one of claims 1-4, wherein the ferredoxin protein comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, and 10.

6. The genetically modified cell of any one of claims 1-5, wherein the heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid is selected from CrtE, CarRP, CarB, and / or CrtZ, optionally wherein the CrtZ gene is not Saccharolobus solfataricus CrtZ (SsCrtZ).

7. The genetically modified cell of any one of claims 1-6, wherein the cell is capable of producing an increased level of carotenoid when cultured in a culture medium comprising an iron source, optionally wherein the iron source is iron sulfate.

8. The genetically modified cell of any one of claims 1-7, wherein the carotenoid is zeaxanthin or a derivative thereof, optionally wherein the derivative of zeaxanthin is not violaxanthin.

9. A genetically modified cell comprising a transgene that encodes a native ferredoxin protein, wherein the genetically modified cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the native ferredoxin protein, optionally wherein the genetically modified cell is a genetically modified microbe.

10. A genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the ferredoxin protein is not YAH1 or ferredoxin-3, optionally wherein the genetically modified cell is a genetically modified microbe, further optionally wherein the ferredoxin protein is selected from APD1, ATM1, ILV3, and NAR1.

11. A genetically modified cell comprising a transgene that encodes a ferredoxin protein, wherein the cell is capable of producing an increased level of a carotenoid compared to a cell having the same genetic background but without the transgene encoding the ferredoxin protein, wherein the carotenoid is not violaxanthin, optionally wherein the genetically modified cell is a genetically modified microbe, further optionally wherein the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin.

12. A method of producing a carotenoid, comprising culturing the genetically modified cell of any one of claims 1-11 in a culture medium, optionally wherein the culture medium comprises an iron source, further optionally wherein the iron source is iron sulfate.

13. A method of making a genetically modified cell of any one of claims 1-11, comprising contacting the cell with a nucleic acid encoding a ferredoxin protein, optionally wherein the nucleic acid is an expression vector.

14. A method of increasing a carotenoid production in a cell, comprising contacting the cell with an agent that increases gene copy number, expression, and / or activity of a ferredoxin, wherein the ferredoxin is not YAH1 or ferredoxin-3, optionally wherein the genetically modified cell is a genetically modified microbe, further optionally wherein the ferredoxin is selected from APD1, ATM1, ILV3, and NAR1.

15. A method of increasing production of a carotenoid in a cell, wherein the carotenoid is not violaxanthin, comprising contacting the cell with an agent that increases gene copynumber, expression, and / or activity of ferredoxin, optionally wherein the genetically modified cell is a genetically modified microbe, further optionally wherein the carotenoid is zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, or beta-cryptoxanthin.

16. The method of claim 14 or 15, wherein the agent is a nucleic acid, a protein, and / or a small molecule, optionally wherein the agent is a nucleic acid encoding a ferredoxin protein.

17. A genetically modified cell comprising (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a transgene that encodes a heterologous carotenoid binding protein (CBP), wherein the transgene enhances the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof.

18. A genetically modified cell comprising (i) a heterologous gene encoding a carotenoid biosynthetic enzyme for producing a carotenoid, and (ii) a genetic modification capable of increasing the expression of a carotenoid binding protein (CBP), wherein the genetic modification enhances the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof.

19. The genetically modified cell of claim 17 or 18, wherein the genetically modified cell is a genetically modified microbe, optionally wherein the genetically modified microbe is a bacterium or a yeast.

20. The genetically modified cell of any one of claims 17-19, wherein the CBP is a wildtype CBP or an engineered CBP.

21. The genetically modified cell of any one of claims 17-20, wherein the CBP is selected from the steroidogenic acute regulatory lipid transfer (START) protein family or the Aster family of proteins, optionally wherein the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog, or human Aster-A or a human Aster-A homolog.

22. The genetically modified cell of any one of claims 17-21, wherein the CBP comprises an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 12 or 14.

23. The genetically modified cell of any one of claims 17-21, wherein the zeaxanthin or a derivative thereof, or astaxanthin or a derivative thereof is selected from zeaxanthin, astaxanthin, adonirubin, canthaxanthin, adonixanthin, and beta-cryptoxanthin.

24. A genetically modified cell comprising a transgene that encodes a heterologous carotenoid binding protein (CBP), wherein the genetically modified cell is capable of producing an increased level of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof, compared to a cell having the same genetic background but without the transgene encoding the heterologous carotenoid binding protein (CBP), optionally wherein the genetically modified cell is a genetically modified microbe.

25. A genetically modified cell comprising a transgene that encodes a heterologous carotenoid binding protein (CBP), wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof, optionally wherein the genetically modified cell is a genetically modified microbe, further optionally wherein the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog, or human Aster-A or a human Aster-A homolog.

26. A method of producing (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof, comprising culturing the genetically modified cell of any one of claims 17- 25 in a culture medium, optionally wherein the culture medium comprises an iron source, further optionally wherein the iron source is iron sulfate.

27. A method of making a genetically modified cell of any one of claims 17-25, comprising contacting the cell with a nucleic acid encoding a heterologous carotenoid binding protein (CBP), optionally wherein the nucleic acid is an expression vector.

28. A method of increasing the production of (X) zeaxanthin or a derivative thereof, or (Y) astaxanthin or a derivative thereof in a cell, comprising contacting the cell with an agent that increases gene copy number, expression, and / or activity of a heterologous carotenoid binding protein (CBP), wherein the CBP is not human lipid binding / transfer protein saposin B (hSapB), supernatant protein factor (SPF), H. gammarus crustacyanin A2 subunit (HgCRA2), or human apolipoprotein B (HsApoB), or any variant thereof, optionally whereinthe genetically modified cell is a genetically modified microbe, further optionally wherein the CBP is Bombyx mori CBP or a Bombyx mori CBP ortholog, or human Aster-A or a human Aster-A homolog.

29. The method of claim 27 or 28, wherein the agent is a nucleic acid, a protein, and / or a small molecule, optionally wherein the agent is a nucleic acid encoding a heterologous CBP.

Images